Treatment of disease by inducing cell apoptosis

ABSTRACT

The present invention relates generally to the treatment and prevention of diseases characterized by excess cell proliferation and/or activation. In particular, the present invention provides compositions and methods to suppress the activation and/or proliferation of various cells. In preferred embodiments, the present invention provides compositions and methods to suppress the activation and/or proliferation of mesenchymally derived cells (including, but not limited to hepatic stellate cells), as well as cells with abnormal growth characteristics. In particularly preferred embodiments, the present invention provides compositions and methods to induce fibrosis.

The present application is a U.S. national entry of International Appln. No. PCT/US01/51123, filed Oct. 26, 2001, which claims priority benefit to U.S. Provisional Patent Appln. Ser. No. 60/244,018 filed Oct. 27, 2000.

This invention was made with government support under DK 38652 and DK 46971, awarded by the National Institutes of Health. The government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates generally to the treatment and prevention of diseases characterized by excess cell proliferation and/or activation. In particular, the present invention provides compositions and methods to suppress the activation and/or proliferation of various cells. In preferred embodiments, the present invention provides compositions and methods to suppress the activation and/or proliferation of mesenchymally derived cells (including, but not limited to hepatic stellate cells), as well as cells with abnormal growth characteristics. In particularly preferred embodiments, the present invention provides compositions and methods to inhibit or eliminate fibrosis. In alternative preferred embodiments, the present invention provides compositions and methods to induce fibrosis.

BACKGROUND OF THE INVENTION

Abnormal cell proliferation is a hallmark of various pathological conditions. In particular, uncontrolled cell growth is associated with diseases such as fibrosis (e.g., hepatic fibrosis), and various other diseases and conditions associated with abnormal cell proliferation.

For example, chronic hepatitis is characterized as an inflammatory liver disease continuing for at least six months without improvement. Chronic hepatitis C represents one form of chronic hepatitis. Left unchecked, chronic hepatitis C can progress to cirrhosis and extensive necrosis of the liver. Although chronic hepatitis C is often associated with deposition of collagen type I leading to hepatic fibrosis, the mechanisms of fibrogenesis remain unknown. Indeed, there is no established treatment for hepatic fibrogenesis related to the over-production of collagen type I (See, e.g., Maher and McGuire, J. Clin. Invest. 86:1641-48 (1990); Chojkier Pathogenesis of hepatic fibrosis. In Extracellular Matrix, Marcel Dekker Inc., New York, N.Y., pp. 541-57 [1993]). However, it is known that ribosomal protein S-6 kinases (RSKs) are critical for survival of cells such as the hepatic stellate cells that overproduce the fibrous tissue that results in cirrhosis. Nonetheless, despite the tremendous research effort and funding dedicated fibrotic diseases (e.g., hepatic fibrosis), there remains a need in the art for compositions and methods that are effective in suppressing the activation and/or proliferation of abnormal cells.

SUMMARY OF THE INVENTION

The present invention relates generally to the treatment and prevention of diseases characterized by excess cell proliferation and/or activation. In particular, the present invention provides compositions and methods to suppress the activation and/or proliferation of various cells, including but not limited to mesenchymal cells. In preferred embodiments, the present invention provides compositions and methods to suppress the activation and/or proliferation of mesenchymally derived cells (including, but not limited to hepatic stellate cells), as well as cells with abnormal growth characteristics. In particularly preferred embodiments, the present invention provides compositions and methods to inhibit or eliminate fibrosis. In alternative preferred embodiments, the present invention provides compositions and methods to induce fibrosis.

The present invention also provides methods and compositions suitable for the suppression of cell activation and/or proliferation. In some preferred embodiments, the present invention also provides methods and compositions suitable for the suppression of hepatic stellate cell activation and/or proliferation. In particularly preferred embodiments, the present invention provides methods for administering C/EBPβ with a mutation of Thr²¹⁷ to Ala. In other embodiments, the endogenous phosphopeptidases associate with caspases 1 and 8 and result in the inhibition of their activation. In alternative embodiments, the mutant Ala²¹⁷ peptides compete with the wild-type peptides, allowing the activation of caspases, resulting in apoptosis.

The modified C/EBPβ peptides of the present invention find use with various tissues and cells. It is contemplated that any suitable route of administration will find use with the present invention. Thus, in some embodiments, the peptides are administered using genetic therapy methods, selective peptide delivery systems, or any other suitable method for delivery to the site of interest. In particularly preferred embodiments, the peptides are delivered to hepatic stellate cells. In alternative preferred embodiments, the peptides are administered parenterally while in still further embodiments, the peptides are administered orally or topically.

In some embodiments, the composition(s) of the present invention is/are administered to the subject in a single dose, while in other embodiments, the composition is administered to the subject in multiple doses. In preferred embodiments, the administering is selected from the group consisting of subcutaneous injection, oral administration, intravenous administration, intraarterial administration, intraperitoneal administration, rectal administration, vaginal administration, topical administration, intramuscular administration, intranasal administration, intrapulmonary administration (e.g., inhalation, insufflation, etc.), intratracheal administration, epidermal administration, transdermal administration, subconjunctival administration, intraocular administration, periocular administration, retrobulbar administration, subretinal administration, suprachoroidal administration, intramedullar administration, intracranial administration, intraventricular administration, and intrathecal administration. In alternative embodiments, the administering is administration from a source selected from the group consisting of mechanical reservoirs, devices, implants, and patches. In still further embodiments, the composition is in a form selected from the group consisting of pills, capsules, liquids, gels, powders, suppositories, suspensions, creams, jellies, aerosol sprays, and dietary supplements. Additionally, the peptides may be administered as an ointment, lotion or gel (i.e., for the treatment of skin and mucosal areas). In some embodiments, it is expected that cells in several tissues will contain an expression vector and express the gene of interest (i.e., such that the peptide(s) of interest are expressed in the tissue(s)).

In alternative embodiments, once the peptides are incorporated into cells, the peptides compete with endogenous C/EBP wild-type protein. In still further embodiments, the presence of mutant C/EBPβ peptide(s) results in the induction of stellate cells apoptosis. In other embodiments, the activation and proliferation of hepatic stellate cells is prevented. In particularly preferred embodiments, this prevention of activation and proliferation of hepatic stellate cells results in decreased or complete cessation of fibrous tissue production in the liver. In some embodiments, the methods are used to treat subjects suffering from or suspected of suffering from chronic liver disease (e.g., including but not limited to hepatitis C, hepatitis B, alcoholism, toxic and genetic liver diseases). Furthermore, it is contemplated that the methods of the present invention will find use in the prevention of liver fibrosis associated with liver rejection following liver transplantation.

The present invention provides modified C/EBPβ peptides from various species. In some preferred embodiments, the murine C/EBPβ peptide comprises a mutation at amino acid 217. In some embodiments, the threonine present in wild-type murine C/EBPβ at position 217 (SEQ ID NO:2) is replaced with alanine (SEQ ID NO:3), while in other embodiments, it is replaced with glutamic acid (SEQ ID NO:4). In alternative preferred embodiments, the rat C/EBPβ peptide comprises a mutation at amino acid 105. In some embodiments, the serine at position 105 of the wild-type rat C/EBPβ (SEQ ID NO:10) is replaced with alanine (SEQ ID NO:11), while in other embodiments, it is replaced with aspartic acid (SEQ ID NO:12). In still further preferred embodiments, the human C/EBPβ comprises a mutation at amino acid 266. In some embodiments, the threonine at position 266 of the wild-type human C/EBPβ (SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:19, and SEQ ID NO:23) is replaced with alanine (SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:20, and SEQ ID NO:24), while in other embodiments, it is replaced with glutamic acid (SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:21, and SEQ ID NO:25). Other mutations in C/EBPβ peptides from various species are contemplated by the present invention. The only requirement of these modified C/EBPβ peptides is that they are capable of inducing or prevent apoptosis in a cell or tissue of interest. In particularly preferred embodiments, the induction of apoptosis results in the reduction (i.e., diminution), elimination, and/or prevention of fibrosis in a cell or tissue of interest. In alternative preferred embodiments, the modified C/EBPβ peptides induce fibrosis (e.g., to promote wound healing).

In some preferred embodiments, the present invention provides modified CCAAT/Enhancer binding proteins capable of inducing apoptosis. In some preferred embodiments, the modified CCAAT/Enhancer binding protein is selected from the group consisting of modified human CCAAT/Enhancer binding proteins, and modified mouse CCAAT/Enhancer binding proteins. In further preferred embodiments, the protein is encoded by an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:24, and SEQ ID NO:25.

The present invention also provides methods for inducing apoptosis comprising the steps of administering at least one modified CCAAT/Enhancer binding protein to at least one cell. In some preferred embodiments, the cell is a mesenchymal cell. In further embodiments, the cell is selected from the group consisting of hepatic cells, lung cells, kidney cells, skin cells, muscle cells, heart cells, glial cells, ocular cells, and vascular cells. In some particularly preferred embodiments, the administration prevents fibrosis. In alternative preferred embodiments, the administration ameloriates fibrosis.

The present invention also provides methods for inducing apoptosis comprising administering the CCAAT/Enhancer binding protein to a subject under conditions such that the endogenous phosphopeptides of the subject inhibit the activation of at least one caspase of the subject. In some preferred embodiments, the administration results in the apoptosis of selected cells within the subject.

The present invention further provides methods for inducing apoptosis comprising administering at least a portion of a modified CCAAT/Enhancer binding protein to at least one cell, wherein the modified CCAAT/Enhancer binding protein is selected from the group consisting of modified murine, modified rat, and modified human CCAAT/Enhancer binding proteins. In some preferred embodiments, the modified murine CCAAT/Enhancer binding protein comprises a mutation at amino acid position 217, wherein the amino acid at position 217 is selected from the group consisting of alanine and glutamic acid. In other preferred embodiments, the modified murine CCAAT/Enhancer binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4. In additional embodiments, the modified human CCAAT/Enhancer binding protein comprises a mutation at amino acid position 266, wherein the amino acid at position 266 is selected from the group consisting of alanine and glutamic acid. In further embodiments, the modified human CCAAT/Enhancer binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:11. In yet additional embodiments, the modified rat CCAAT/Enhancer binding protein comprises a mutation at amino acid position 105, wherein the amino acid at position 105 is selected from the group consisting of alanine and aspartic acid. In further embodiments, the modified rat CCAAT/Enhancer binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:14 and SEQ ID NO:15. In some preferred embodiments, apoptosis is induced in at least one cell selected from the group consisting of hepatic cells, heart cells, lung cells, kidney cells, ocular cells, neural cells, muscle cells, epithelial cells, endothelial cells, mesenchymal cells, and skin cells. In some preferred embodiments, the cell(s) is/are within a subject. In further embodiments, the subject is a mammal. In some particularly preferred embodiments, the mammal is a human. In additional embodiments, the human is suffering from a fibrosis-related disease, wherein the fibrosis-related disease is selected from the group consisting of hepatic disease, brain damage, myocardial infarction, arteriosclerosis, ocular fibrosis, fibrotic skin conditions, and fibrotic pulmonary disease.

The present invention also provides methods for inducing fibrosis comprising the administration of at least a portion of a modified CCAAT/Enhancer binding protein to at least one tissue, wherein the modified CCAAT/Enhancer binding protein is selected from the group consisting of modified murine, modified rat and modified human CCAAT/Enhancer binding proteins. In some preferred embodiments, prior to the administration of CCAAT/Enhancer binding protein, at least one tissue exhibits impaired wound healing. In some embodiments, the induction of fibrosis provides improved wound healing in at least one tissue. In some particularly preferred embodiments, the modified CCAAT/Enhancer binding protein is the amino acid sequence set forth in SEQ ID NO:4.

Definitions

To facilitate understanding of the invention that follows, a number of terms and phrases are defined below.

As used herein, the term “mesenchyme” refers to the embryonic tissue that is the origin of connective tissue, as well as muscle, blood cells and vessels, epithelium, and some glands.

As used herein, the term “gene” means the deoxyribonucleotide sequences comprising the coding region of a structural gene and including sequences located adjacent to the coding region on both the 5′ and 3′ ends for a distance of several kb on either end such that the gene corresponds to the length of the full-length mRNA. The sequences which are located 5′ of the coding region and which are present on the mRNA are referred to as 5′ non-translated sequences. The sequences which are located 3′ or downstream of the coding region and which are present on the mRNA are referred to as 3′ non-translated sequences. A genomic form or clone of a gene contains coding sequences, termed exons, alternating with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences.” Introns are segments of a gene which are transcribed into heterogenous nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5′ and 3′ end of the sequences which are present on the RNA transcript. These sequences are referred to as “flanking” sequences or regions (these flanking sequences are located 5′ or 3′ to the non-translated sequences present on the mRNA transcript). The 5′ flanking region may contain regulatory sequences such as promoters and enhancers which control or influence the transcription of the gene. The 3′ flanking region may contain sequences which direct the termination of transcription, posttranscriptional cleavage and polyadenylation.

As used herein the term “coding region” when used in reference to a structural gene refers to the nucleotide sequences which encode the amino acids found in the nascent polypeptide as a result of translation of an mRNA molecule. The coding region is bounded, in eukaryotes, on the 5′ side by the nucleotide triplet “ATG” which encodes the initiator methionine and on the 3′ side by one of the three triplets which specify stop codons (i.e., TAA, TAG, TGA).

As used herein, the term “structural gene” refers to a DNA sequence coding for RNA or a protein. In contrast, “regulatory genes” are structural genes which encode products (e.g., transcription factors) which control the expression of other genes.

As used herein, the term “regulatory element” refers to a genetic element which controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, enhancer elements, etc. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (Maniatis et al., Science 236:1237 [1987]). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells and viruses (analogous control elements [i.e., promoters], are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types (for review see Voss, et al., Trends Biochem. Sci., 11:287 [1986]; and Maniatis, et al., Science 236:1237 [1987]). For example, the SV40 early gene enhancer is very active in a wide variety of cell types from many mammalian species and has been widely used for the expression of proteins in mammalian cells (Dijkema, et al., EMBO J. 4:761 [1985]). Other examples of promoter/enhancer elements active in a broad range of mammalian cell types are those from the human elongation factor 1α gene (Uetsuki et al., J. Biol. Chem., 264:5791 [1989]; Kim et al., Gene 91:217 [1990]; and Mizushima and Nagata, Nucl. Acids. Res., 18:5322 ([990]) and the long terminal repeats of the Rous sarcoma virus (Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777 [1982]) and the human cytomegalovirus (Boshart et al., Cell 41:521 [1985]).

The terms “promoter element” or “promoter” as used herein refer to a DNA sequence that is located at the 5′ end of (i.e., precedes) a gene in a DNA polymer and provides a site for initiation of the transcription of the gene into mRNA.

The terms “gene of interest” and “nucleotide sequence of interest” refer to any gene or nucleotide sequence, respectively, the manipulation of which may be deemed desirable for any reason by one of ordinary skill in the art.

The term “expression vector” as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

A “modification” as used herein in reference to a nucleic acid sequence refers to any change in the structure of the nucleic acid sequence. Changes in the structure of a nucleic acid sequence include changes in the covalent and non-covalent bonds in the nucleic acid sequence. Illustrative of these changes are mutations, mismatches, strand breaks, as well as covalent and non-covalent interactions between a nucleic acid sequence (which contains unmodified and/or modified nucleic acids) and other molecules. Illustrative of a covalent interaction between a nucleic acid sequence and another molecule are changes to a nucleotide base (e.g., formation of thymine glycol) and covalent cross-links between double-stranded DNA sequences which are introduced by, for example, ultraviolet radiation or by cis-platinum. Yet another example of a covalent interaction between a nucleic acid sequence and another molecule includes covalent binding of two nucleic acid sequences to psoralen following ultraviolet irradiation. Non-covalent interactions between a nucleic acid sequence and another molecule include non-covalent interactions of a nucleic acid sequence with a molecule other than a nucleic acid sequence and other than a polypeptide sequence. Non-covalent interactions between a nucleic acid sequence with a molecule other than a nucleic acid sequence and other than a polypeptide sequence are illustrated by non-covalent intercalation of ethidium bromide or of psoralen between the two strands of a double-stranded deoxyribonucleic acid sequence. The present invention contemplates modifications which cause changes in a functional property (or properties), such changes manifesting themselves at a variety of time points.

The term “allelic series” when made in reference to a gene refers to wild-type sequences of the gene. An “allelic series of modifications” as used herein in reference to a gene refers to two or more nucleic acid sequences of the gene, where each of the two or more nucleic acid sequences of the gene contains at least one modification when compared to the wild-type sequences of the gene.

As used herein, the term “mutation” refers to a deletion, insertion, or substitution. A “deletion” is defined as a change in a nucleic acid sequence in which one or more nucleotides is absent. An “insertion” or “addition” is that change in a nucleic acid sequence which has resulted in the addition of one or more nucleotides. A “substitution” results from the replacement of one or more nucleotides by a molecule which is a different molecule from the replaced one or more nucleotides. For example, a nucleic acid may be replaced by a different nucleic acid as exemplified by replacement of a thymine by a cytosine, adenine, guanine, or uridine. Alternatively, a nucleic acid may be replaced by a modified nucleic acid as exemplified by replacement of a thymine by thymine glycol.

The term “mismatch” refers to a non-covalent interaction between two nucleic acids, each nucleic acid residing on a different polynucleic acid sequence, which does not follow the base-pairing rules. For example, for the partially complementary sequences 5′-AGT-3′ and 5′-AAT-3′, a G-A mismatch is present.

The terms “nucleic acid” and “unmodified nucleic acid” as used herein refer to any one of the known four deoxyribonucleic acid bases (i.e., guanine, adenine, cytosine, and thymine). The term “modified nucleic acid” refers to a nucleic acid whose structure is altered relative to the structure of the unmodified nucleic acid. Illustrative of such modifications would be replacement covalent modifications of the bases, such as alkylation of amino and ring nitrogens as well as saturation of double bonds.

The term “modified cell” refers to a cell which contains at least one modification in the cell's genomic sequence.

The term “nucleic acid sequence-modifying agent” refers to an agent which is capable of introducing at least one modification into a nucleic acid sequence. Nucleic acid sequence-modifying agents include, but are not limited to, chemical compounds (e.g., N-ethyl-N-nitrosurea (ENU), methylnitrosourea (MNU), procarbazine hydrochloride (PRC), triethylene melamine (TEM), acrylamide monomer (AA), chlorambucil (CHL), melphalan (MLP), cyclophosphamide (CPP), diethyl sulfate (DES), ethyl methane sulfonate (EMS), methyl methane sulfonate (MMS), 6-mercaptopurine (6 MP), mitomycin-C (MMC), procarbazine (PRC), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ³H₂O, and urethane (UR)), and electromagnetic radiation (e.g., X-ray radiation, gamma-radiation, ultraviolet light).

The term “wild-type” when made in reference to a gene refers to a gene which has the characteristics of that gene when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the gene. In contrast, the term “modified” or “mutant” refers to a gene or gene product which displays modifications in sequence and/or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product. Indeed, in some preferred embodiments of the present invention, the modified gene products (i.e., peptides and proteins) induce apoptosis or fibrosis. Thus, in some particularly preferred embodiments, the modified C/EBPβs of the present invention are suitable for the induction of apoptosis in fibrotic tissue, such that the detrimental effects of fibrosis are prevented, reduced, or eliminated. In further embodiments, the modified C/EBPβs of the present invention find use in the induction of fibrosis, such that processes that depend upon fibrosis (e.g., wound healing) are promoted.

A “variant of C/EBPβ” is defined as an amino acid sequence which differs by one or more amino acids from the C/EBPβ wild-type sequence. Variant C/EBPβs also comprise “modified C/EBPβ” proteins and peptides. In some embodiments, variant peptides (i.e., modified) may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant (i.e., modified) peptide has “nonconservative” changes (e.g., replacement of a glycine with a tryptophan). Similar minor variations also include amino acid deletions or insertions (i.e., additions), or both. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, DNAStar software. In some particularly preferred embodiments, the threonine in position 217 of the wild-type murine C/EBPβ is substituted with an alanine or glutamic acid. In still further embodiments, the serine in position 105 of the wild-type rat C/EBPβ is substituted with an alanine or aspartic acid. In yet further embodiments, the threonine in position 266 of the wild-type human C/EBPβ is substituted with either an alanine or glutamic acid.

As used herein, the term “biological agent” refers to an agent that is useful for diagnosing or imaging or that can act on a cell, organ or organism, including but not limited to drugs (e.g., pharmaceuticals) to create a change in the functioning of the cell, organ or organism.

As used herein, the term “sample” is used in its broadest sense. In one sense it can refer to a composition comprising a therapeutic agent. In another sense, it is Qmeant to include a specimen or culture obtained from any source. Biological samples may be obtained from animals (including humans) and encompass tissues (e.g., biopsy samples), fluids, solids, tissues, and gases. Biological samples also include blood products, such as plasma, serum and the like. These examples are not to be construed as limiting the sample types applicable to the present invention.

As used herein, the term “chemotherapeutic agent” refers to an agent that inhibits the growth or decreases the survival of neoplastic or pathogenic microbial cells or inhibits the propagation (which includes without limitation replication, viral assembly or cellular infection) of a virus. It is intended that the definitions encompass compounds purified from the natural source, as well as compounds prepared synthetically or by semisynthesis. Thus, it is not intended that the present invention be limited to these compounds produced in by a particular method or from any specific source.

The term “compound” refers to any chemical entity, pharmaceutical, drug, and the like that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function. Compounds comprise both known and potential therapeutic compounds. A compound can be determined to be therapeutic by screening using the screening methods of the present invention. A “known therapeutic compound” refers to a therapeutic compound that has been shown (e.g., through animal trials or prior experience with administration to humans) to be effective in such treatment. In other words, a known therapeutic compound is not limited to a compound efficacious in the treatment of cancer.

The term “test compound” refers to any chemical entity, pharmaceutical, drug, and the like that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function. Test compounds comprise both known and potential therapeutic compounds. A test compound can be determined to be therapeutic by screening using the screening methods of the present invention. A “known therapeutic compound” refers to a therapeutic compound that has been shown (e.g., through animal trials or prior experience with administration to humans) to be effective in such treatment or prevention. In other words, a known therapeutic compound is not limited to a compound efficacious in the treatment of fibrosis.

A compound is said to be “in a form suitable for administration to the mammal” when the compound may be administered to a mammal by any desired route (e.g., oral, intravenous, subcutaneous, intramuscular, etc.) and the compound or its active metabolites appears in the blood of the mammal. Administration of a compound to a pregnant female may result in delivery of the compound to the fetuses of the pregnant animal.

A “composition comprising a given polynucleotide sequence” as used herein refers broadly to any composition containing the given polynucleotide sequence. The composition may comprise an aqueous solution. Compositions comprising polynucleotide sequences encoding human, mouse, and/or rat C/EBPβ or fragments thereof may be employed as hybridization probes. In this case, the CEBPβ-encoding polynucleotide sequences are typically employed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., SDS) and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).

As used herein, the term “pharmaceutical composition” refers to any composition or compound suitable for administration to a subject for treatment and/or prevention of disease. It is contemplated that such compositions encompass drugs that are effective in reducing or eliminating signs and symptoms of disease. It is not intended that the present invention be limited to any particular dosage or administration strategy. It is also intended that the term encompass various regimens, including, but not limited to regimens in which a combination of pharmaceutical compositions are provided in separate vehicles (e.g., separate tablets, pills, liquids, etc.).

As used herein, the term “therapeutically effective amount” refers an amount of a therapeutic agent that inhibits the growth or decreases the survival of neoplastic cells.

As used herein, the terms “subtherapeutic amount” and “subtherapeutic concentration” refer to an amount of a therapeutic that is administered at a concentration that would not be expected to exhibit complete destruction of cells.

As used herein, the term “anti-proliferative” refers to a therapeutic agent that suppresses or inhibits the growth of abnormal cells, but does not necessarily kill the cells.

As used herein, the term “cytotoxic” drug or agent refers to a therapeutic agent useful in treating disease that results in the death of cells. In some preferred embodiments, cytotoxic drugs are particularly effective against rapidly dividing cells.

As used herein, the term “IC₅₀” refers to the concentration at which 50% cytotoxicity is obtained. Cytotoxicity can be measured by the method of Alley et al., Cancer Res. 48: 589-601, 1988 or Scudiero et al., Cancer Res., 48:4827, 1988, or by any other suitable method known in the art. In one embodiment, cytotoxicity can be measured based on the drug concentration at which a 50% reduction in the activity of mitochondrial enzymes is observed.

As used herein, the term “subject” refers to a human or other animal of interest. In some preferred embodiments, the subject is any living animal that is capable of being treated with a pharmaceutical composition.

As used herein, the term “hepatitis C” refers to subjects infected with the hepatitis C virus, a single-stranded RNA virus that possesses a lipid-containing envelope and is thought to be a member of the flavivirus family. The term encompasses all forms of hepatitis C, including acute hepatitis C and all forms of chronic hepatitis C (e.g., chronic active hepatitis and chronic persistent hepatitis).

As used herein, the phrase “symptoms of hepatitis C” refers broadly to clinical manifestations, laboratory and imaging results, as well as liver morphology and histology exhibited by subjects which suggest the presence of hepatitis C. Clinical manifestations may include, but are not limited to, abdominal pain, jaundice, hepatosplenomegaly, and ascites. Laboratory and imaging results may include, but are not limited to, elevated serum aminotransferase, bilirubin, and gamma-globulin levels, as well as an enlarged liver on computed tomography, magnetic resonance imaging, and hepatic ultrasonography. Hepatic morphological and histological indicators of hepatitis C may include, but are not limited to, deposition of fibrotic tissue evident through liver biopsy.

As used herein, the term “fibrosis” refers to the formation of fibrous tissue as a reparative or reactive process, such as that which occurs during the development of scar tissue which replaces normal tissue following injury, inflammation, and/or infection. In some embodiments, the term refers to the replacement of normal smooth muscle or other normal tissue with fibrous connective tissue. Fibrosis can occur in any tissue and involve localized or more widespread effects. Localized fibrosis may be complicated by infarctions, abscesses, bronchiectasis, and/or other pathologies.

As used herein, the term “apoptosis” refers to cell death. In particular, apoptosis refers to programmed cell death which is distinct from the necrotic process. During apoptosis, cells undergo various changes that result in the eventual lysis of the cell into apoptotic bodies which are then typically phagocytosed by other cells.

As used herein, the phrases “symptoms indicating fibrosis,” refer to the morphological and histological indicators of fibrosis in any tissue. In particularly preferred embodiments, the term “hepatic fibrosis” refers to morphological and histological indicators of fibrosis involving the liver. Such indicators may include, but are not limited to, deposition of fibrotic tissue evident through liver biopsy and activation of the fibrogenesis cascade as evidenced by increased MDA-adducts, stellate cell activation, and enhanced expression of c-myb and collagen α1(I) mRNA in stellate cells.

As used herein, the term “diminished” means that there has been a reduction in the extent of the symptoms of fibrosis, etc. In general, such a reduction is demonstrated by objective indicators. For example, comparison of liver biopsy samples taken before and after administration of a therapeutic agent may indicate a reduction in hepatic fibrosis. In addition, reduction of symptoms may also be demonstrated by subjective indicators, such as a reduction in abdominal pain.

As used herein, the term “therapeutic composition” refers to a composition that includes a compound in a pharmaceutically acceptable form that prevents and/or reduces fibrosis, including but not limited to hepatic fibrosis. The characteristics of the form of the therapeutic composition will depend on a number of factors, including the mode of administration. The therapeutic composition may contain diluents, adjuvants and excipients, among other things.

As used herein, the term “parenterally” refers to administration to a subject through some means other than through the gastrointestinal tract or the lungs. Common modes of parenteral administration include, but are not limited to, intravenous, intramuscular, and subcutaneous administration.

As used herein, the terms “therapeutic amount,” “effective amount,” and the like, refer to that amount of a compound or preparation that successfully prevents the symptoms of fibrosis and/or reduces the severity of symptoms. The effective amount of a therapeutic composition may depend on a number of factors, including the age, immune status, race, and sex of the subject and the severity of the fibrotic condition and other factors responsible for biologic variability.

The “non-human animals having a genetically engineered genotype” of the invention are preferably produced by experimental manipulation of the genome of the germline of the non-human animal. These genetically engineered non-human animals may be produced by several methods including the introduction of a “transgene” comprising nucleic acid (usually DNA) into an embryonal target cell or integration into a chromosome of the somatic and/or germ line cells of a non-human animal by way of human intervention, such as by the methods described herein. Non-human animals which contain a transgene are referred to as “transgenic non-human animals”. A transgenic animal is an animal whose genome has been altered by the introduction of a transgene.

As used herein, the term “transgene” refers to a foreign gene that is placed into an organism by introducing the foreign gene into newly fertilized eggs or early embryos. The term “foreign gene” refers to any nucleic acid (e.g., gene sequence) which is introduced into the genome of an animal by experimental manipulations and may include gene sequences found in that animal so long as the introduced gene does not reside in the same location as does the naturally-occurring gene.

As used herein, “non-human mammal expressing mutant C/EBP protein” is a mammal which expresses a C/EBP protein that is different from that found in a wild-type mammal. A mammal that “lacks the ability to produce functional C/EBP” is a mammal that produces undetectable levels of C/EBP (i.e., a level which is not statistically above background levels in the assay employed). A functional C/EBP protein is a C/EBP, which has the same properties as does the wild-type C/EBP including molecular weight. A functionally active fragment of human C/EBP is capable of functioning in vitro and/or in vivo in a manner that is similar or the same as full-length C/EBP. “Functional activity” also encompasses such activities normally associated with C/EBP proteins.

DESCRIPTION OF THE DRAWINGS

FIG. 1 provides data showing that phosphorylation of C/EBPβ on Thr²¹⁷ is required for hepatic stellate cell survival. Panel A provides phosphopeptide maps of in vivo labeled endogenous C/EBPβ in day-4 quiescent and activated primary mouse stellate cells cultured from the time of isolation on EHS or collagen type I matrix, respectively. The arrow indicates the peptide containing Phospho-Thr²¹⁷ in activated cells. These results indicate that collagen type I induces phosphorylation of C/EBPβ on Thr²¹⁷. Panel B provides phosphopeptide maps of in vitro labeled C/EPBβ. Active p90RSK phosphorylated C/EPBβ, but not C/EBPβ-Ala²¹⁷ on Thr²¹⁷ in vitro (arrowheads). Panel C provides results for cells cultured on collagen type I transfected with MSV-C/EBPβ antisense (AS), MSV-C/EBPβ sense (S), or MSV-C/EBPβ-Ala²¹⁷ together with CMV-GFP (green fluorescent protein) and p90RSK (1 μg each), as indicated in the Figure. Four hours after transfection, annexin-V-PE binding to plasma membranes was determined. The values presented are the percentage of transfected cells (green) that were annexin-V-PE (+) (red) (P<0.05 for C/EBPβ AS, and C/EBPβ-Ala²¹⁷). Panel D provides results for cells transfected as in Panel A with vectors expressing dominant negative mutants for MEKK1, IκBα, or p90RSK, or treated with the proteasome inhibitor lactacystin (10 μM) (closed bars), and co-transfected with MSV-C/EBPβ-Glu²¹⁷ (1 μg each) (open bars) as indicated. Annexin-V-PE binding was determined as described for Panel A (P<0.05 for C/EBPβ-Glu²¹⁷). Panel E provides results for cells transfected as described for Panel C, with a dominant-negative p90RSK, treated with FAS (100 nM for 6 hours) or cultured in a serum-free medium for 6 hours (closed bars), and co-transfected with MSV-C/EBPβ-Glu²¹⁷ (1 μg each) (open bars), as indicated. Cell death was determined by nuclear staining with Hoechst 33342 (P<0.05 for C/EBPβ-Glu²¹⁷ in RSK mut and FAS-treated cells).

FIG. 2 provides results showing increased apoptosis in hepatic stellate cells from C/EBPβ^(−/−) and C/EBPβ-Ala²¹⁷ transgenic mice. Panel A shows results from primary stellate cells isolated from C/EBPβ^(+/+), C/EBPβ^(−/−) and C/EBPβ-Ala²¹⁷ mice and cultured on a collagen type I matrix. Six hours after cultivation, an annexin-V-PE binding assay was performed (P<0.05 for C/EBPβ^(−/−) and C/EBPβ-Ala²¹⁷). Panel B shows results for nuclear staining of cells described above (Panel A) with Hoechst 33342. In addition, cells from C/EBPβ-Ala²¹⁷ mice were treated with a cell permeant caspase 8 inhibitor (IETD) (1.05 nM) or transfected with MSV-C/EBPβ, C/EBPβ-Ala²¹⁷, C/EBPβ-Ala²¹⁰, or RSK (1 μg each), as indicated (P<0.05 for C/EBPβ^(−/−), C/EBPβ-Ala²¹⁷, C/EBPβ-Ala²¹⁷+ RSK+C/EBPβ-Ala²¹⁰. Panel C provides data showing representative examples of annexin-V and mitochondrial sensor and Hoechst 33342 assays in primary stellate cells described above for Panels A and B. As indicated, stellate cells from C/EBPβ-Ala²¹⁷ mice displayed increased annexin-V-PE binding and altered mitochondrial sensor assay results, and loss of nuclear integrity. Apoptotic cells are indicated with arrowheads.

FIG. 3 provides results showing that C/EBPβ-Thr²¹⁷ associates with procaspases 1 and 8. Panel A provides results showing that stellate cells, identified by confocal microscopy with glial fibrillary acidic protein (GFAP) displayed active caspase 3 in livers from C/EBPβ-Ala²¹⁷, but not C/EBPβ^(+/+), mice 12 h after treatment with CCl₄. Colocalization of GFAP and active caspase 3 is indicated by the arrows. Asterisks indicate sinusoidal lumens in C/EBPβ^(+/+) mice. Only background (i.e., red) staining was observed when the GFAP antibody was omitted. Mineral oil induced no changes. Panel B provides data showing increased p90RSK association with C/EBPβ. These results are for an RSK immunoblot performed on C/EBPβ^(+/+), C/EBPβ-Ala²¹⁷, and C/EBPβ^(−/−) (control) immunoprecipitates from protein lysates (500 μg) from samples described for Panel A. RSK association was increased in stellate cells isolated from C/EBPβ^(+/+) and C/EBPβ-Ala²¹⁷ mice treated with CCl₄. C/EBPβ-PThr²¹⁷ association with RSK and active RSK-PSer³⁸⁰ (in RSK immunoprecipitates) was increased in C/EBPβ^(+/+) and C/EBPβ-Ala²¹⁷ mice treated with CCl₄. Panel C provides results showing that association of procaspases 1 and 8 with C/EBPβ and C/EBPβ-PThr²¹⁷ (in C/EBPβ immunoprecipitates) was increased in C/EBPβ^(+/+) mice treated with CCl₄. Reciprocal immunoprecipitates confirmed these associations. Panel D provides results showing that the activities of caspases 1 and 8 are increased in stellate cell lysates from C/EBPβ^(−/−) and C/EBPβ-Ala²¹⁷, but not C/EBPβ^(+/+) mice treated with CCl₄, as described above for Panel A. Caspase activity was determined by the release of the chromogenic substrate using 5×10⁶ cells per point. The values are presented as the mean±one half of the range of duplicates for caspase 1 (empty bars) and caspase 8 (closed bars) activities in CCl₄-treated animals (corrected for the basal activities in untreated animals).

FIG. 4 provides results showing that C/EBPβ PThr²¹⁷ associates with procaspases 1 and 8, and inhibits their activation. Panel A provides schematic representations of C/EBPβ peptides. The activation (dotted bars), DNA binding (solid bars) and dimerization (hatched bars) domains are shown. Panel B shows results for stellate cells transfected with the indicated plasmids together with CMV-GFP (0.3 μg each) and cultured on a collagen type I matrix (P<0.05 for C/EBPβ 216-256-Ala²¹⁷ and RSK mutant). Panel C provides results showing that C/EBPβ and procaspase 8 were detected in activated stellate cells isolated from C/EBPβ^(+/+) mice by confocal microscopy using specific antibodies (Santa Cruz Biotechnology). Control C/EBPβ^(−/−) stellate cells were negative for C/EBPβ, positive for procaspase 8, and apoptotic. Panel D provides immunoblot results for MSV vectors expressing C/EBPβ-HA (hemagglutinin protein) peptides (0.3 μg) transfected into activated stellate cells. The expressed peptides were immunoprecipitated with anti-HA antibodies. C/EBPβ-PThr²¹⁷ was detected using a specific antibody, only in cells expressing wild-type C/EBPβ. Immunoblots showed a greater association of procaspases 1 and 8 with C/EBPβ-PThr²¹⁷ and C/EBPβ peptides containing Glu²¹⁷ than C/EBPβ peptides containing Ala²¹⁷. RSK was associated with all C/EBPβ peptides. Neither C/EBPβ nor procaspases 1 and 8 were detected in C/EBPβ immunoprecipitates from cells expressing C/EBPβ AS. Reciprocal immunoprecipitations of procaspases and RSK confirmed these associations.

FIG. 5 provides results showing the direct association of C/EBPβ-Glu²¹⁷ with procaspases 1 and 8. Panel A provides an immunoblot showing results indicating that purified recombinant C/EBPβ-Glu²¹⁷ and C/EBPβ 216-253-Glu²¹⁷ (200 μM) co-immunoprecipitated purified recombinant procaspases 1 (0.6 U) and 8 (0.12 μg) 7- and 12-fold more than C/EBPβ-HA-Ala²¹⁷ and C/EBPβ 216-253-HA-Ala²¹⁷, as quantitated by densitometry. Panel B provides results showing that purified C/EBPβ-HA-Glu²¹⁷ and C/EBPβ 216-219-PThr²¹⁷ and -Glu²¹⁷ synthetic tetrapeptides inhibited the in vitro self-activation of caspases 1 and 8. Enzyme activity was measured in the absence (control) or presence of C/EBPβ peptides (200 μM) for 2 h, using a procaspases 1 (1U) (not shown) and 8 (0.2 μg) activity assay. Values are presented as the mean±one half the range of duplicates. Panel C provides results for cells cultured on a collagen type I matrix and incubated with the indicated cell permeant, purified synthetic N-acetyl, C-aldehyde C/EBPβ tetra-peptides (10 μM) or with recombinant C/EBPβ-HA peptides as indicated, together with the Chariot reagent (Active Motif) for 4 h. Control cells were incubated without peptides (P<0.05 for all peptides containing Ala²¹⁷).

FIG. 6 provides results showing endogenous C/EBPβ is phosphorylated on Ser¹⁰⁵ in rat stellate cells. Panel A provides phosphopeptide maps performed in day-4 quiescent and activated primary rat hepatic stellate cells cultured as described above for FIG. 1. These results show that collagen type I induces phosphorylation of rat C/EBPβ on Ser¹⁰⁵. The arrow indicates the peptide containing Phospho-Ser¹⁰⁵ in activated cells. Panel B provides phosphopeptide maps of in vitro labelled rC/EBPβ. Active p90RSK phosphorylated rC/EBPβ in vitro, but not rC/EBPβ-Ala¹⁰⁵ on Ser¹⁰⁵ (indicated by arrowheads).

FIG. 7 provides data showing that phosphorylation of rat C/EBPβ on Ser¹⁰⁵ prevents stellate cell apoptosis. Panel A provides results for mouse stellate cells transfected as described herein (See, FIG. 2), with mutant p90RSK or treated with lactacystin (10 μM) (closed bars), and co-transfected with CMV-rC/EBPβ-Asp¹⁰⁵ (1 μg each) (open bars) as indicated. Annexin-V-PE binding was determined as described for FIG. 2 (P<0.05 for rC/EBPβ-Asp¹⁰⁵). Panel B provides results of an annexin-V-PE assay for stellate cells from C/EBPβ^(+/+) (closed circles) and rC/EBPβ-Asp¹⁰⁵ transgenic (open circles) mice treated with lactacystin (0-30 μM). The annexin-V-PE assay was performed 6 hours after treatment began (P<0.05 for rC/EBPβ-Asp¹⁰⁵ transgenic mice for 5-25 μM lactacystin). Panel C provides results for rC/EBPβ-HA constructs (0.3 μg) transfected into rat stellate cells. The expressed peptides were immunoprecipitated with anti-HA antibodies. Using a specific antibody, rCEBPβ-PSer¹⁰⁵ was detected only in cells that expressed wild-type rC/EBPβ. An immunoblot for procaspases 1 and 8 showed a greater association with C/EBPβ-PSer¹⁰⁵ and rC/EBPβ-Asp¹⁰⁵ than with C/EBPβ-Ala¹⁰⁵ peptides. Reciprocal immunoprecipitations confirmed these associations. Panel D provides results for stellate cells isolated from C/EBPβ−/− mice incubated with the indicated cell permeant, purified synthetic N-acetyl-C-aldehyde C/EBPβ tetrapeptides (10 μM) for 4 hours together with the Chariot reagent. Control cells were incubated without peptides (P<0.05, for both tetrapeptides). Panel E provides results for Ac-KE²¹⁷VD-CHO and Ac-KKPD¹⁰⁵-CHO synthetic tetrapeptides (200 μM). These results indicated that these tetrapeptides inhibited the in vitro self-activation of caspase 8. Enzyme activity was measured using a colorimetric substrate as described in the Examples. Baseline caspase 8 activity was 4.7 U (100%). Values are presented as the mean±one half of the range of duplicates.

FIG. 8 provides the nucleotide sequence (SEQ ID NO:1) and amino acid sequence (SEQ ID NO:2) of wild-type mouse C/EBPβ.

FIG. 9 provides the amino acid sequence of modified mouse C/EBPβ in which the threonine present in wild-type murine C/EBPβ at position 217 (SEQ ID NO:2) is replaced with alanine (SEQ ID NO:3), as well as the amino acid sequence of modified mouse C/EBPβ in which the threonine present in wild-type murine C/EBPβ at position 217 (SEQ ID NO:2) is replaced with glutamic acid (SEQ ID NO:4).

FIG. 10 provides the nucleotide sequence (SEQ ID NO:5) and amino acid sequence (SEQ ID NO:6) of wild-type human C/EBP.delta., which corresponds to Genbank Accession No. M83667.

FIG. 11 provides the amino acid sequence of modified human C/EBP.delta., in which the threonine at position 265 is replaced with alanine (SEQ ID NO:7), as well as the amino acid sequence of modified human C/EBP.delta., in which the threonine at position 265 is replaced with glutamic acid (SEQ ID NO:8).

FIG. 12 provides the amino acid sequence (SEQ ID NO:9) of human c/EBP.beta., as well as the amino acid sequence of modified human C/EBP.beta., in which the threonine at position 266 of human C/EBP.beta. (SEQ ID NO:9) is replaced with alanine (SEQ ID NO:10), and the amino acid sequence of modified human C/EBP.beta., in which the threonine at position 266 of human C/EBP.beta. (SEQ ID NO:9) is replaced with glutamic acid (SEQ ID NO:11).

FIG. 13 provides the nucleotide sequence (SEQ ID NO:18) and amino acid sequence (SEQ ID NO:19) of wild-type human C/EBPβ that corresponds to Genbank Accession No. X52560.

FIG. 14 provides the amino acid sequence of modified human C/EBPβ, in which the threonine at position 266 of the wild-type human C/EBPβ (SEQ ID NO:19) is replaced with alanine (SEQ ID NO:20), as well as the amino acid sequence of modified human C/EBPβ, in which the threonine at position 266 of the wild-type human C/EBPβ (SEQ ID NO:19) is replaced with glutamic acid (SEQ ID NO:21).

FIG. 15 provides the nucleotide sequence (SEQ ID NO:22) and amino acid sequence (SEQ ID NO:23) of wild-type human C/EBPβ that corresponds to Genbank Accession No. NM_(—)005194.

FIG. 16 provides the amino acid sequence of modified human C/EBPβ, in which the threonine at position 266 of the wild-type human C/EBPβ (SEQ ID NO:23) is replaced with alanine (SEQ ID NO:24), as well as the amino acid sequence of modified human C/EBPβ, in which the threonine at position 266 of the wild-type human C/EBPβ (SEQ ID NO:23) is replaced with glutamic acid (SEQ ID NO:25).

FIG. 17 provides the nucleotide sequence (SEQ ID NO:12) and amino acid sequence (SEQ ID NO:13) of wild-type rat c/EBPβ.

FIG. 18 provides the amino acid sequence of modified rat C/EBPβ in which the serine at position 105 of the wild-type rat C/EBPβ (SEQ ID NO:13) is replaced with alanine (SEQ ID NO:14), as well as the amino acid sequence of modified rat C/EBPβ in which the serine at position 105 of the wild-type rat C/EBPβ (SEQ ID NO:13) is replaced with aspartic acid (SEQ ID NO:15).

DESCRIPTION OF THE INVENTION

The present invention relates generally to the treatment and prevention of diseases characterized by excess cell proliferation and/or activation. In particular, the present invention provides compositions and methods to suppress the activation and/or proliferation of various cells. In preferred embodiments, the present invention provides compositions and methods to suppress the activation and/or proliferation of mesenchymally derived cells (including, but not limited to hepatic stellate cells), as well as cells with abnormal growth characteristics. In particularly preferred embodiments, the present invention provides compositions and methods to inhibit or eliminate fibrosis. In alternative preferred embodiments, the present invention provides compositions and methods to induce fibrosis.

The CCAAT/Enhancer Binding Protein β (C/EBPβ) (Descombes et al., Genes Dev., 4:1541-1551 [1990]; and Akira et al., EMBO J., 9:1897-1906 [1990]) mediates the proliferative effects of oxidative stress and of ribosomal protein S-6 kinase (RSK) activated by transforming growth factor (TGF) α in colonic cancer cells (Chinery et al., Nat. Med., 3:1233-1241 [1997]) and primary mouse hepatocytes (Buck et al., Mol. Cell., 4:1087-1092 [1999]), respectively. However, the mechanisms linking C/EBPβ (LAP, NF-IL6) to cell proliferation are unknown. Indeed, an understanding of these mechanisms is not necessary in order to use the present invention.

Activation of the ERK/MAPK signal transduction pathway promotes cell proliferation through several mechanisms, including stimulation of nucleotide synthesis, gene expression, protein synthesis and cell growth (Whitmarsh and Davis, J. Mol. Med., 74:589-607 [2000]). Many of these MAPK's roles in cell growth are mediated by p90RSK (Nebreda and Gavin, Science 286:1309-1310 [1999]). RSK phosphorylates and inactivates the pro-apoptotic protein BAD (Bonni et al., Science 286:1358-1362 [1999]); up-regulates transcription of the anti-apoptotic gene Bcl-2 through phosphorylation and activation of CREB (Bonni et al., supra); and facilitates gene expression by inducing chromatin remodeling via phosphorylation of histone H3 (Sassone-Corsi et al., Science 285:886-891 [1999]).

In response to epidermal growth factor, the ERK/MAPK cascade regulates the de novo synthesis of pyrimidine nucleotides by activating carbamoyl phosphate synthetase II (Graves et al., Nature 403:328-332 [2000]). Other mechanisms by which the ERK/MAPK signaling pathway modulates cell survival and the cell cycle (Whitmarsh and Davis, J. Mol. Med., 403:255-256 [1996]) include: a) activation of transcription by phosphorylation of transcriptional factors (Whitmarsh and Davis, J. Mol. Med., 74:589-607 [1996]; Bhatt and Ferrell, Science 286:1362-1365 [1999]) and histone H3 and HMG-14 (Sassone-Corsi et al., supra; and Thomson et al., EMBO J., 17:4779-4793 [1999]); b) stimulation of translation through phosphorylation of initiation factor 4E (eIF-4E) (Pyronnet et al., EMBO J., 18:270-279 [1999]); and c) promotion of DNA replication by increasing expression of cyclin D1 (Lavoie et al., J. Biol. Chem., 271:206086-20616 [1996]). RSK, which is activated by ERK/MAPK phosphorylation, plays an essential role in the ERK/MAPK signaling pathway regulating cell survival and the cell cycle (Bonni et al., supra; Bhatt and Ferrell, supra; Gross et al., Science 286:1365-1367 [1999]; and Sassone-Corsi et al., supra). Thus, the function of C/EBPβ in cell survival mediated by RSK was of interest in the development of the present invention.

Primary mouse hepatic stellate cells were used during the development of the present invention, as overproduction of fibrous tissue by these cells (Friedman et al., Proc. Natl. Acad. Sci. USA 82:8681-8685 [1985]; and Ankoma-Sey and Friedman, in Strain and Diehl (eds), Liver Growth and Repair, Chapman & Hall, London, pages 512-537 [1998]) is a critical step in the development of liver cirrhosis following liver injury (Chojkier, in Strain and Diehl (eds.) Liver Growth and Repair, supra, at pages 430-450). Furthermore, these cells remain quiescent, as in the normal liver (Lee et al., J. Clin. Invest., 96:2461-2468 [1995]; and Ankoma-Sey and Friedman, supra), when cultured on an EHS (Matrigel) matrix but are rapidly activated and proliferate with the induction of oxidative stress by either collagen type I or TGFα in culture (Lee et al., supra; and Friedman et al., J. Biol. Chem., 264:10756-10762), which also modulate C/EBPβ's effects on cell growth (Chinery et al., supra; and Buck et al., [1999], supra). Similarly, stellate cell activation follows the stimulation of oxidative stress both in experimental liver injury with CCl₄ (Houglum et al., J. Clin. Invest., 96:2269-2276 [1995]) and in human liver diseases induced by alcohol, genetic hemochromatosis, porphyria and viral hepatitis (Chojkier, supra). However, until the development of the present invention, it was unknown that the survival dependency of activated hepatic stellate cells requires phosphorylation of C/EBPβ (e.g., mouse C/EBPβ; (m) C/EBPβ) on Thr²¹⁷ by RSK. As described in greater detail herein, the hepatotoxin CCl₄ induced activation of RSK, phosphorylation of C/EBPβ on Thr²¹⁷ and proliferation of stellate cells in normal mice, but caused apoptosis of these cells in C/EBPβ^(−/−) and C/EBPβ-Ala²¹⁷ (a dominant negative non-phosphorylatable mutant) transgenic mice. In other words, following the induction of hepatic oxidative stress with CCl₄, stellate cells from C/EBPβ^(−/−) or C/EBPβ-Ala²¹⁷ (a non-phosphorylatable mutant) transgenic, but not C/EBPβ^(+/+), mice developed apoptosis. Furthermore, both C/EBPβ-PThr²¹⁷ and the phosphorylation mimic C/EBPβ-Glu²¹⁷, but not C/EBPβ-Ala²¹⁷, were found to associate with procaspases 1 and 8 in vitro and in vivo, and inhibit their activation. The data obtained during the development of the present invention indicate that C/EBPβ phosphorylation of Thr²¹⁷ creates a functional XEXD caspase substrate/inhibitor box (KPhospho-T²¹⁷VD) that is mimicked by C/EBPβ-Glu²¹⁷ (KE²¹⁷VD). Consistent with this observation, C/EBPβ^(−/−) and C/EBP-Ala²¹⁷ stellate cells were rescued from apoptosis by either the cell permeant KE²¹⁷VD tetrapeptide or C/EBPβ-Glu²¹⁷, as described in greater detail herein.

Results obtained during the development of the present invention and described in further detail herein, indicate that there is a novel C/EBPβ mechanism for cell survival downstream of RSK, preventing activation of procaspases 1 and 8 (Thornberry and Lazebnik, Science 281:1312-1316 [1998]). These caspases activate downstream effector procaspases (Earnshaw et al., Ann. Rev. Biochem., 68:383-424 [1999]). Physiologically relevant signaling pathways in stellate cells, such as CCl₄ in mice and collagen type I in culture (Friedman et al., J. Biol. Chem., 264:10756-10762 [1989]; and Rudolph et al., Science 287:1253-1258 [2000]), result in the activation of RSK and phosphorylation of endogenous C/EBPβ on Thr²¹⁷. C/EBPβ-PThr²¹⁷, but not unphosphorylated C/EBPβ, associates with procaspases 1 and 8 (as detected by immunofluorescence, co-immunoprecipitation and direct in vitro association of recombinant proteins) and inhibits their processing, which blocks the apoptotic cascades and allows survival of stellate cells. Thus, the present data provide the first demonstration that phosphorylation of a transcription factor by the ERK/MAPK/RSK pathway stimulates its association with procaspases, preventing their activation. Although it is possible that phosphorylation of C/EBPβ on Thr²¹⁷ to create a functional XEXD caspase substrate/inhibitor box (Thornberry et al., J. Biol. Chem., 272:17907-17911 [1997]; and Blanchard et al., J. Mol. Biol., 302:9-16 [2000]) explains the anti-apoptotic role of C/EBPβ, structural analysis is required to elucidate the exact mechanism. Regardless, an understanding of the mechanisms is not necessary in order to use the present invention.

The inhibition of procaspase 1 and 8 activation by C/EBPβ-PThr²¹⁷ occurs under conditions in which C/EBPβ expression is physiologically induced, such as activation of normal stellate cells either in mice treated with the hepatotoxin CCl₄ or in culture on a collagen type I matrix. Moreover, apoptosis is induced under these conditions in stellate cells from C/EBPβ^(−/−) mice. These experiments indicate that the findings are not the spurious result of overexpressing C/EBPβ. Expression of C/EBPβ-Ala²¹⁷ in transgenic mice also induced apoptosis of stellate cells upon exposure to growth stimuli (CCl₄ in mice and collagen in culture). In contrast, the phosphorylation mimic C/EBPβ-Glu²¹⁷ mutant rescued cells from apoptosis induced either by expressing MEKK1, IKBα or RSK dominant negative mutants or by treating the cells with the proteasome inhibitor lactacystin (Chen et al., Nature 374:386-388 [1995b]; Nakajima et al., Cell 86:465-474 [1996]; and Lee et al., Cell 88:213-222 [1997]). Expression of C/EBPβ-Glu²¹⁷ in stellate cells also prevented FAS-induced apoptosis, which is mediated by activation of procaspase 8 (Ashkenazi and Dixit, Science 281:1305-1208 [1998]), but not apoptosis induced by serum deprivation, which is mediated by activation of procaspase 9 (Joza et al., Nature 410:549-554 [2001]). These results indicate selective effects of C/EBPβ-PThr²¹⁷ on apoptosis. C/EBPβ's activation and dimerization domains are not necessary for its association with or inhibition of procaspases 1 and 8. Furthermore, preliminary experiments in stellate cells using reporter genes, which contain C/EBPβ binding domains (Descombes et al. supra; and Houglum et al., J. Clin. Invest., 94:808-814 [1994]), did not show differences in transcription activation between C/EBPβ wild type and the phosphorylation or Ala²¹⁹ mutants.

C/EBPβ-PThr²¹⁷ is mimicked by C/EBPβ-Glu²¹⁷ (KE²¹⁷VD) and therefore, conforms to the current knowledge about the structural requirements for caspase inhibitory/substrate tetrapeptides. These tetrapeptides require an aspartic acid (D) residue at the P1 position (Thornberry and Lazebnik, supra), which is also present in C/EBPβ at amino acid D²¹⁹ (Cao et al., Genes Develop., 5:1538-1552 [1991]). Using a positional scanning substrate library, it has been shown that the optimal sequence for caspase 8 is I/L/V (P4 position) EXD, although I, V, W, T, P and D are also acceptable at the P4 position, by virtue of being downstream cleavage sites in the apoptotic cascade (Thornberry et al., supra). These findings have been recently corroborated by crystallographic studies of caspase 8. (Blanchard et al., supra). Although group III caspases (including caspase 8) have a preference for small hydrophobic residues at P4, kinetic and crystallographic studies have shown that DEVD, a specific group II inhibitor, containing a hydrophilic residue at this position, interacts favorably with the enzyme in subsite S4, and it is an almost equally potent inhibitor as the specific group III inhibitor IETD (Blanchard et al., supra). The K²¹⁶ in C/EBPβ has a similar hydrophobicity as D (Boyle et al., Meth. Enzymol., 201:110-149 [1991]). In addition, Blanchard et al. argue that subsite S3 of caspase 8, which interacts with position P3 (but not with P4) of the tetrapeptide, is crucial for determining the specificity, and that the original classification of caspases need to be revised, especially for caspase 8 (Blanchard et al., supra). Like the selective effects of C/EBPβ-PThr²¹⁷ observed during the development of the present invention, CrmA inhibits procaspases 1 and 8 selectively compared to procaspases 3, 6 and 7 (Zhou et al., J. Biol. Chem., 272:7797-7800 [1997]). However, other reports have suggested that CrmA also inhibits caspases 4, 5, 9 and 10 (Garcia-Calvo et al., J. Biol. Chem., 273:32608-32613 [1998]). In addition, the baculovirus p35 is a potent, albeit less selective, inhibitor of procaspases 1, 3, 6, 8, and 10 (Andrade et al., Immun., 8:451-460 [1998]). In addition, experiments conducted during the development of the present invention have shown that the sequence K-Phospho-T²¹⁷VD (or KE²¹⁷VD) within C/EBPβ is effective in associating with procaspases 1 and 8 and blocking their activation.

The experiments with C/EBPβ^(−/−) cells and with dominant negative Ala²¹⁷ and dominant positive Glu²¹⁷ mutants, including C/EBPβ-Ala²¹⁷ transgenic mice, strongly support the present invention. The C/EBPβ-Ala²¹⁷ mutant associates with RSK and acts as a dominant negative, following proliferation of stellate cells in animals and in culture induced by CCl₄ and collagen type I, respectively. The synthetic Ac-KA²¹⁷VD-CHO peptide stimulates apoptosis of stellate cells, resembling the dominant negative effects of C/EBPβ-Ala²¹⁷ and C/EBPβ216-253-Ala²¹⁷. These effects could result from the inhibition of RSK and/or the facilitation of procaspases 1 and 8 activation, possibly by impeding the binding of C/EBPβ-PThr²¹⁷ to procaspases 1 and 8, while allowing their self-cleavage. Nonetheless, an understanding of the mechanisms is not necessary in order to use the present invention.

Although rC/EBPβ has a naturally occurring Ala for Thr substitution at position 217, rC/EBPβ also has a Ser for Ala substitution at position 105 (Buck et al., [1999], supra). It was also observed that endogenous rC/EBPβ is phosphorylated on Ser¹⁰⁵ (the RSK rat phosphoacceptor homologue). Expression of the dominant positive rC/EBPβ-Asp¹⁰⁵ was sufficient to rescue cells from apoptosis induced by expressing a dominant negative mutant RSK or by treatment with a proteasome inhibitor. Activation of the PKCα or MAPK signaling pathways results in phosphorylation of rC/EBPβ on Ser¹⁰⁵ (Trautwein et al., Nature 364:544-547 [1993]; and Buck et al. [1999], supra). Moreover, stellate cells isolated from rC/EBPβ-Asp¹⁰⁵ transgenic mice were refractory to the induction of apoptosis by lactacystin. As described for C/EBPβ, rC/EBPβ peptides containing either P-Ser¹⁰⁵ or its phosphorylation mimic Asp¹⁰⁵, associated with procaspases 1 and 8 in rat stellate cells. The Ac-KKPD¹⁰⁵-CHO tetrapeptide also inhibited activation of procaspase 8. These data indicate that although C/EBPβ or rC/EBPβ are phosphorylated on different amino acid residues, both phosphorylated proteins are sufficient to rescue cells from apoptosis mediated by caspase 8.

In contrast, the non-phosphorylatable rC/EBPβ-Ala¹⁰⁵ mutant behaves as a dominant negative (comparable to the C/EBPβ-Ala²¹⁷ mutant), inducing apoptosis of both mouse and rat stellate cells. The rC/EBPβ-PSer¹⁰⁵ sequence is mimicked by KKPD¹⁰⁵, which contains the indispensable D at the P1 position and the highly preferred P at the P2 position as a substrate/inhibitor (XXPD) for granzyme B, as determined by using synthetic substrate libraries (Harris et al., J. Biol. Chem., 273:27364-27373 [1998]). The initiator caspases, caspase 8 and granzyme B, share the tetrapeptide IETD sequence of procaspase 3 as a substrate/inhibitor (Thornberry et al., 1997, supra; Harris et al., supra), indicating that caspase 8 may also recognize the XXPD substrate. This was confirmed by the experimental data obtained during the development of the present invention. The tetrapeptide Ac-KKPD¹⁰⁵-CHO (rC/EBPβ) significantly inhibited apoptosis of stellate cells from C/EBPβ^(−/−) mice.

The data obtained during the development of the present invention are relevant to diseases that result from the activation of mesenchymal cells, which leads to excessive tissue repair mechanisms (e.g., brain gliosis, liver cirrhosis, and lung and kidney fibrosis). The results described herein indicate that the nonphosphorylatable tetrapeptides of the C/EBPβ RSK phosphoacceptor are suitable for therapeutic uses, as they induce apoptosis of various cells following their activation. Thus, for hepatic stellate cells, these tetrapeptides find use in the prevention of the development of liver fibrosis and cirrhosis. Indeed, C/EBPβ-Ala²¹⁷ transgenic mice are refractory to the induction of liver fibrosis and cirrhosis following the chronic administration of CCl₄. In addition, because IL-1 has been implicated in the pathogenesis of acute neurodegeneration (Rothwell et al., J. Clin. Invest., 100:2648-2652 ([1997]), inhibition of caspase 1 activation and its processing of the IL-1 precursor is contemplated to ameliorate diseases associated with fibrosis and/or apoptosis.

The creation of a functional XEXD caspase inhibitory box by phosphorylation reported herein, may be a prevalent biological mechanism through evolution. Indeed, potential functional XEXD boxes that would be created upon phosphorylation of threonine (Phospho-TVD, analogous to EVD) were identified in more than 22,000 sites and of serine (EV-Phospho-S, analogous to EVD) in more than 27,000 sites in a database containing ˜350,000 proteins. If only 1% of these sequences is phosphorylated in vivo, this would generate functional XEXD caspase inhibitory boxes in ˜500 proteins. However, as indicated herein, an understanding of the mechanisms involved is not necessary in order to use the present invention.

As discussed in greater detail herein, in one embodiment of the present invention, polynucleotide sequences or fragments thereof which encode C/EBPβ or fusion proteins or functional equivalents thereof, may be used in recombinant DNA molecules to direct expression of C/EBPβ in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and these sequences may be used to clone and express C/EBPβ.

As will be understood by those of skill in the art, it may be advantageous to produce C/EBPβ nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.

The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter C/EBPβ-encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, or introduce mutations, and so forth. In another embodiment of the present invention, natural, modified, or recombinant nucleic acid sequences encoding C/EBPβ may be ligated to a heterologous sequence to encode a fusion protein. For example, to screen peptide libraries for inhibitors of human C/EBPβ activity, it may be useful to encode a chimeric (e.g., human) C/EBPβ protein that can be recognized by a commercially available antibody. A fusion protein may also be engineered to contain a cleavage site located between the C/EBPβ encoding sequence and the heterologous protein sequence, so that the C/EBPβ may be cleaved and purified away from the heterologous moiety.

In another embodiment of the present invention, sequences encoding C/EBPβ may be synthesized, in whole or in part, using chemical methods well known in the art (See e.g., Caruthers et al. Nucl. Acids Res. Symp. Ser., 215-223 [1980]; and Horn et al., Nucl. Acids Res. Symp. Ser., 225-232 [1980]). Alternatively, the protein itself may be produced using chemical methods to synthesize the amino acid sequence of C/EBPβ, or a portion thereof. For example, peptide synthesis can be performed using various solid-phase techniques (Roberge et al., Science 269:202-204 [1995]) and automated synthesis may be achieved, for example, using commercially available synthesizers. These synthesized peptide may be substantially purified by preparative high performance liquid chromatography (HPLC), using any suitable method known in the art. The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure). Additionally, the amino acid sequence of C/EBPβ or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.

As described herein in greater detail, in order to express a biologically active C/EBPβ the nucleotide sequences encoding C/EBPβ or functional equivalents, may be inserted into appropriate expression vector (i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence). Methods well known to those skilled in the art find use in the construction of expression vectors containing sequences encoding C/EBPβ and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. A variety of expression vector/host systems may be utilized to contain and express sequences encoding C/EBPβ. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Control elements and regulatory sequences are also contemplated, as appropriate.

In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a “prepro” form of the protein may also be used to facilitate correct insertion, folding and/or function. Different host cells known in the art which have specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express C/EBPβ may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type. Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase, adenine phosphoribosyltransferase, and various other suitable systems known in the art. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection. Although the presence/absence of marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed. For example, if the sequence encoding C/EBPβ is inserted within a marker gene sequence, recombinant cells containing sequences encoding human C/EBPβ can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding C/EBPβ under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequence encoding and express C/EBPβ may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein.

A variety of protocols for detecting and measuring the expression of human C/EBPβ, using either polyclonal or monoclonal antibodies specific for the protein known in the art find use in the present invention. Examples include enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).

A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding C/EBPβ include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternatively, the sequences encoding C/EBPβ or any portions thereof may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kit. Suitable reporter molecules or labels, which may be used, include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding C/EBPβ may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode C/EBPβ may be designed to contain signal sequences which direct secretion of portions of C/EBPβ through a prokaryotic or eukaryotic cell membrane. Other recombinant constructions may be used to join sequences encoding C/EBPβ to nucleotide sequences encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.). The inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, Calif.) between the purification domain and human C/EBPβ may be used to facilitate purification.

In addition to recombinant production, fragments of C/EBPβ may be produced by direct peptide synthesis using solid-phase techniques that are well known in the art (See e.g., Merrifield, J. Am. Chem. Soc., 85:2149-2154 [1963]). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved by any commercially available synthesizer suitable for the project. In addition, various fragments of C/EBPβ may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention relates generally to the treatment and prevention of diseases characterized by excess cell proliferation and/or activation. In particular, the present invention provides compositions and methods to suppress the activation and/or proliferation of various cells. In preferred embodiments, the present invention provides compositions and methods to suppress the activation and/or proliferation of mesenchymally derived cells (including, but not limited to hepatic stellate cells), as well as cells with abnormal growth characteristics. In particularly preferred embodiments, the present invention provides compositions and methods to inhibit or eliminate fibrosis. In alternative preferred embodiments, the present invention provides compositions and methods to induce fibrosis.

Phosphorylation of C/EBPβ on Thr²¹⁷ is Required for Hepatic Cell Survival

To test whether collagen type I matrix induces C/EBPβ phosphorylation, primary mouse hepatic stellate cells were labeled for 12 hr with ³²P-orthophosphate and cultured either on EHS (Matrigel) (quiescent cells) or collagen type I (activated cells), as described in Example 5. Phosphopeptide mapping of immunoprecipitated C/EBPβ showed that the collagen type I matrix induced a site-specific phosphorylation of endogenous C/EBPβ on Thr²¹⁷, a p90RSK phosphoacceptor domain (Buck et al., [1999], supra), and on another, yet unidentified, phosphoacceptor site (See, FIG. 1, Panels A and B). C/EBPβ phosphorylation was negligible in quiescent mouse stellate cells cultured on an EHS matrix (See, FIG. 1, Panel A).

Next, the functional relevance of C/EBPβ and its phosphorylation on Thr²¹⁷ were ascertained. Activated primary mouse hepatic stellate cells were transfected with either wild-type (sense) or antisense C/EBPβ expressing vectors. Stellate cells expressing C/EBPβ antisense, but not sense, RNA (Buck et al., EMBO J., 13:851-860 [1994]) lacked detectable C/EBPβ protein and did not proliferate, as detected by the expression of proliferating cell nuclear antigen (Bravo et al., Nature 326:515-517 [1987]; Buck et al., [1994], supra; and Buck et al., [1999], supra). However, these cells displayed increased annexin-V binding to phosphatidylserine in plasma membranes, an early indicator of apoptosis (Rudel and Bokoch, supra) (See, FIG. 1, Panel C).

To determine whether phosphorylation of mouse C/EBPβ on Thr²¹⁷ (i.e., C/EBPβ-PThr²¹⁷) is critical for survival, mouse stellate cells were transfected with a MSV expression vector for the nonphosphorylatable C/EBPβ-Ala²¹⁷ mutant. Transfected cells were identified by the expression of the co-transfected CMV-GFP (green fluorescent protein) using triple-channel fluorescence microscopy, as described in Example 2. Cells expressing the C/EBPβ-Ala²¹⁷ mutant (Buck et al., [1999], supra) did not proliferate and became apoptotic as determined by live microscopy (See, FIG. 1, Panel C) and cell sorting analysis (FACS) for the transfection indicator green fluorescent protein and annexin-V. Cell death was confirmed by nuclear staining with Hoechst 33342. Co-expression of RSK was unable to rescue apoptosis induced by C/EBPβ-Ala²¹⁷ (See, FIG. 1, Panel C).

Conversely, induction of apoptosis in hepatic stellate cells by blocking activation of the antiapoptotic NFκB (Beg and Baltimore, Science 274:782-784 [1996]; and Wang et al., Science 274:784-787 [1996]) or RSK (Buck et al., [1999], supra; Bonni et al., supra; Bhatt and Ferrell, supra; Gross et al., supra; and Sassone-Corsi et al., supra) activity was rescued by the phosphorylation mimic C/EBPβ-Glu²¹⁷ mutant (Buck et al., [1999], supra) (See, FIG. 1, Panel D).

MEK kinase 1 (MEKK1) activates NFRB through phosphorylation of IκB kinases α and β leading to phosphorylation of IκB (Lee et al., Cell 88:213-222 [1997]), which undergoes rapid proteolysis via the ubiquitin-proteasome pathway (Chen et al., Genes Dev., 9:1586-1597 [1995a]). Therefore, stellate cells were transfected with expression vectors for the dominant negative mutants for MEKK1, IκBα and RSK, or treated cells with lactacystin (a proteasome inhibitor) to induce apoptosis. In each case, stellate cell apoptosis was partially inhibited by the C/EBPβ-Glu²¹⁷ mutant (See, FIG. 1, Panel D), indicating that in the absence of active NFκB or RSK, phosphorylation of C/EBPβ on Thr²¹⁷ promotes cell survival. In addition, expression of C/EBPβ-Glu²¹⁷ prevented stellate cell apoptosis induced by the RSK mutant or FAS ligand, but not by serum deprivation (See, FIG. 1, Panel E), indicating that it has a selective effect on caspase activation pathways.

Hepatic Stellate Cells From C/EBPβ^(−/−) and C/EPBβ-Ala²¹⁷ Transgenic Mice Display Increased Apoptosis Following CCl₄ Treatment

To ascertain the functional relevance of mouse C/EBPβ, and its phosphorylation on Thr²¹⁷, the survival of primary hepatic stellate cells isolated from mice with a targeted deletion of C/EBPβ (Buck et al., [1999], supra) was analyzed. C/EBPβ^(−/−), but not C/EBPβ^(+/+), cells became rapidly apoptotic on collagen type I as determined by annexin-V binding using live microscopy (FIG. 2, Panel A). To study the role of C/EBPβ-PThr²¹⁷ on stellate cell survival, mice expressing a nonphosphorylatable dominant negative C/EBPβ-Ala²¹⁷ mutant transgene were developed. Although C/EBPβ-Ala²¹⁷ transgenic mice were developmentally normal, hepatic stellate cells isolated from these animals consistently failed to survive when activated by collagen type I (See, FIG. 2). The apoptotic phenotype of stellate cells isolated from C/EBPβ^(−/−) and C/EBPβ-Ala²¹⁷ transgenic mice was corroborated by using a mitochondrial sensor assay (Green and Reed, Science 281:1309-1312 [1998]), which showed that they had an altered mitochondrial membrane permeability (FIG. 2, Panel C), and by their pycnotic nuclear staining pattern (See, FIG. 2, Panels B and C). Apoptosis of C/EBPβ-Ala²¹⁷ stellate cells was blocked by a cell permeant caspase 8 inhibitor (IETD) or by expression of C/EBPβ-Glu²¹⁷ (See, FIG. 2, Panel B).

Next, the determination was made whether hepatic stellate cell activation is stimulated by CCl₄, a hepatotoxin that induces hepatic oxidative stress and liver fibrogenesis (Houglum et al., supra; and Chojkier, supra). Hepatic stellate cells, identified by their glial fibrillary acidic protein and vimentin staining (Ankoma-Sey and Friedman, supra), were induced to proliferate in the liver of C/EBPβ^(+/+) mice following treatment with a single dose of CCl₄. In contrast, CCl₄ induced stellate cell apoptosis in the livers of C/EBPβ^(−/−) and C/EBPβ-Ala²¹⁷ transgenic mice (See, FIG. 3), as determined by the presence of an active effector caspase 3. Because activated p90RSK is able to phosphorylate C/EBPβ on Thr²¹⁷ (Buck et al., [1999], supra) in vitro, the determination of whether RSK was associated with C/EBPβ in these animals was made.

CCl₄ Treatment Induces Activated RSK, C/EBP-βThr²¹⁷ and its Association with Procaspases 1 and 8 in Hepatic Stellate Cells of Normal Mice

Because activated p90RSK is able to phosphorylate C/EBPβ on Thr²¹⁷ in vitro (FIG. 1, Panel B) (Buck et al., [1999] supra), analyses were conducted to determine whether RSK was associated with C/EBPβ. The results indicated that there was an increased RSK associated with C/EBPβ in stellate cells from C/EBPβ^(+/+) and C/EBPβ-Ala²¹⁷ transgenic mice treated with CCl₄ (FIG. 3, Panel B). Conversely, when RSK was immunoprecipitated with specific antibodies, the level of co-precipitated C/EBPβ was consistently increased in C/EBPβ^(+/+) and C/EBPβ-Ala²¹⁷ transgenic mice treated with CCl₄ (FIG. 3, Panel B). CCl₄ increased the expression and the activity of RSK, as determined with a specific antibody against RSK-PSer³⁸⁰ (FIG. 3, Panel B). In C/EBPβ^(+/+) mice, CCl₄ also induced phosphorylation of endogenous C/EBPβ on Thr²¹⁷, as detected with a newly developed affinity purified antibody specific for this phosphorylated epitope (FIG. 3, Panel B). These findings indicate that phosphorylation of C/EBPβ on Thr²¹⁷ by RSK is indispensable for stellate cell survival. Given that the Thr²¹⁷ phosphoacceptor in C/EBPβ contains a KT²¹⁷VVD sequence (identical to human C/EBPβ) that upon phosphorylation by RSK can be functionally mimicked by the C/EBPβ-Glu²¹⁷ sequence KE²¹⁷VD (Buck et al., [1999], supra), and that the latter is similar to a XEXD box found in inhibitors and substrates of caspases (Thornberry et al., J. Biol. Chem., 272:17907-17911 [1997]; and Blanchard et al., J. Mol. Biol., 302:9-16 [2000]), experiments were conducted to investigate whether C/EBPβ-PThr²¹⁷ associates with procaspases. The results indicated that procaspases 1 and 8 (FIG. 3, Panel C), but not procaspases 3, 7 or 9 (data not shown), in C/EBPβ immunoprecipitates (containing C/EBPβ-PThr²¹⁷) of stellate cells isolated from C/EBPβ^(+/+) mice, following treatment with CCl₄. The levels of procaspases 1 and 8 from C/EBPβ^(+/+) mice treated with CCl₄ were increased, and their immunoprecipitates contained C/EBPβ (FIG. 3, Panel C). The association of C/EBPβ with procaspases 1 and 8 remained baseline in cells of C/EBPβ-Ala²¹⁷ transgenic mice treated with CCl₄ (FIG. 3, Panel C) and in cells of C/EBPβ^(+/+) mice treated with control mineral oil vehicle. In addition, the enzymatic activities of caspases 1 and 8 (FIG. 3, Panel D), and that of the effector caspase 3 (FIG. 3, Panel A), were increased in stellate cells of C/EBPβ^(−/−) or C/EBPβ-Ala²¹⁷, but not of C/EBPβ^(+/+), mice following treatment with CCl₄. These results indicate that phosphorylation of C/EBPβ on Thr²¹⁷ with the creation of a functional XEXD box (or expression of the phosphorylation mimic C/EBPβ-Glu²¹⁷ mutant) is required for C/EBPβ to associate with procaspases 1 and 8. These data also indicate that phosphorylation of C/EBPβ may prevent processing and activation of procaspases 1 and 8, since neither K-Phospho-T²¹⁷VD nor KE²¹⁷VD are the preferred substrates for caspases 1 or 8 (Wilson et al., Nature 370:270-275 [1994]; Margolin et al., J. Biol. Chem., 272:7223-7228 [1997]; Earnshaw et al., Ann. Rev. Biochem., 68:383-424 [1999]), and C/EBPβ does not appear to be cleaved by caspase 1 or 8 (data not shown). In addition, apoptosis of C/EBPβ-Ala²¹⁷ cells was rescued by expressing RSK and C/EBPβ, but not RSK and C/EBPβ-Ala²¹⁹, containing a mutation of the critical P1 Asp residue of the XEXD box (Thornberry and Lazebnik, supra) (See, FIG. 2, Panel B).

C/EBPβ-PThr²¹⁷, Lacking the Activation and Dimerization Domains is Sufficient for Stellate Cell Survival on Collagen Type I

Expression of C/EBPβ 1-253, with a deletion of the dimerization domain (Descombes et al., [1990], supra) (FIG. 4, Panel A) and containing Thr²¹⁷ or Glu²¹⁷, but not Ala²¹⁷, was found to be compatible with cell survival. C/EBPβ 1-215 lacking the DNA binding and dimerization domains and the KT²¹⁷VD phosphoacceptor (FIG. 4, Panel A) had no effect on cell survival. In contrast. C/EBPβ 216-253-Ala²¹⁷, but not Glu²¹⁷, lacking the activation and dimerization domains (FIG. 4, Panel A), induced apoptosis in control cells even when wild type RSK was expressed (FIG. 4, Panel B). In addition, expression of C/EBPβ216-253-Glu²¹⁷ was sufficient to rescue cells from apoptosis induced by a catalytically inactive RSK mutant (RSK N′C′), (Nakajima et al., Cell 86:465-474 [1996]) (FIG. 4, Panel B). In agreement with previous reports (Mao et al., J. Biol. Chem., 273:23621-23624 [1998]; and Ritter et al., Eur. J. Cell Biol., 79:358-364 [2000]), procaspases 1 and 8 were detected predominantly in the cytoplasm of activated stellate cells, which colocalized with C/EBPβ (FIG. 4, Panel C).

Immunoprecipitation of C/EBPβ-HA and C/EBPβ216-253-HA from stellate cells showed that both the P-Thr²¹⁷ and the phosphorylation mimic Glu²¹⁷, co-precipitated with procaspases 1 and 8 more efficiently than the nonphosphorylatable Ala²¹⁷ (FIG. 4, Panel D). Similar results were obtained when the reciprocal immunoprecipitation was performed with procaspases 1 and 8 antibodies. Therefore, neither the activation nor the dimerization domain of C/EBPβ is required for stellate cell survival.

C/EBPβ-PThr²¹⁷ Peptides Associate with Procaspases 1 and 8 and Inhibit Their Activation

In addition, the effect of purified recombinant C/EBPβ peptides on the self-activation of recombinant caspases 1 and 8 in vitro was determined. Consistent with the in vivo results described above, C/EBPβ-HA-Glu²¹⁷ and C/EBPβ216-253-HA-Glu²¹⁷ peptides directly associated with caspases 1 and 8 about 7- and 12-fold more effectively than C/EBPβ-HA-Ala²¹⁷ (FIG. 5, Panel A). Moreover, recombinant C/EBPβ-Glu²¹⁷, and the synthetic tetrapeptides Ac-K-Phospho-T²¹⁷VD-CHO and Ac-KEE²¹⁷VD-CHO directly inhibited the in vitro self-activation of recombinant caspases 1 and 8 (FIG. 5, Panel B). The dominant negative cell permeant peptides Ac-KA²¹⁷VD-CHO consistently induced apoptosis to a degree comparable to C/EBPβ-Ala²¹⁷ and C/EBPβ 216-253-Ala²¹⁷ (FIG. 5, Panel C). In contrast, wild type and Glu²¹⁷ C/EBPβ peptides did not induce apoptosis (FIG. 5, Panel C).

Rat C/EBPβ-PSer¹⁰⁵, the Functional Homologue of Mouse C/EBPβ-PThr²¹⁷, Associates with Procaspases 1 and 8 In Vivo and In Vitro and Inhibit their Activation

Although the C/EBPβ-Thr²¹⁷ phosphoacceptor is highly conserved through evolution, rat (r) C/EBPβ has evolved with a double change, lacking the Thr²¹⁷ phosphoacceptor but having a compensatory Ser¹⁰⁵ RSK phosphoacceptor (Descombes et al., supra). The presence of C/EBPβ-PThr²¹⁷ or rC/EBPβ-PSer¹⁰⁵ in mouse and rat cells, respectively, is critical for the induction of hepatocyte proliferation by TGFα (Buck et al., [1999], supra). Collagen type I induced phosphorylation of endogenous rC/EBPβ on Ser¹⁰⁵ and on other sites in proliferating rat hepatic stellate cells (FIG. 6, Panel A). rC/EBPβ phosphorylation was negligible in quiescent rat stellate cells cultured on an EHS matrix (FIG. 6, Panel A). The phosphopeptide induced by collagen type I had the same mobility as the previously described PSer¹⁰⁵ peptide, a p90RSK phosphoacceptor (Trautwein et al., Nature 364:544-547 [1993]; Buck et al., [1999], supra). Tryptic phosphopeptide analysis of rC/EBPβ demonstrated that p90RSK can phosphorylate C/EBPβ on Ser¹⁰⁵ (FIG. 6, Panel B), as well as other sites; phosphorylation of the rC/EBPβ-Ala¹⁰⁵ mutant did not yield this phosphopeptide (FIG. 6, Panel B), suggesting that this peptide contains Ser¹⁰⁵. Phosphorylation of endogenous rC/EBPβ on Ser¹⁰⁵ was confirmed using specific antibodies against this epitope, as described below.

Additional experiments were conducted to determine whether rC/EBPβ-PSer¹⁰⁵ rescues cells from apoptosis. Stellate cells were induced to develop apoptosis by expressing the dominant negative RSK mutant (Nakajima et al., supra) or by treatment with lactacystin. Expression of the phosphorylation mimic rC/EBPβ-Asp¹⁰⁵ rescued mouse (FIG. 7, Panel A) and rat (data not shown) stellate cells from apoptosis. The nonphosphorylatable rC/EBPβ-Ala¹⁰⁵ mutant, like C/EBPβ-Ala²¹⁷, induced apoptosis of stellate cells (data not shown), since neither mutant has a RSK phosphoacceptor site. These results indicate that rC/EBPβ-PSer¹⁰⁵ (or its phosphorylation mimic rC/EBPβ-Asp¹⁰⁵) is able to block the apoptotic pathway as effectively as C/EBPβ-PThr²¹⁷ (or its phosphorylation mimic C/EBPβ-Glu²¹⁷) (FIG. 1).

Because the rC/EBPβ-Asp¹⁰⁵ mutant behaves as a phosphorylation mimic of rC/EBPβ-PSer¹⁰⁵ in rescuing cells from apoptosis (FIG. 7, Panel A), mice expressing a rC/EBPβ-Asp¹⁰⁵ transgene were developed. Although rC/EBPβ-Asp¹⁰⁵ transgenic mice were developmentally normal, stellate cells isolated from these animals were resistant to the induction of apoptosis by lactacystin (FIG. 7, Panel B). Lactacystin induced a significant and rapid increase in C/EBPβ +/+ stellate cell apoptosis (FIG. 7, Panel B). rC/EBPβ-HA and rC/EBPβ 92-142-HA showed that P-Ser¹⁰⁵ and the phosphorylation mimic Asp¹⁰⁵ coimmunoprecipitated from rat stellate cells with procaspases 1 and 8 more efficiently than the nonphosphorylatable Ala¹⁰⁵ (FIG. 7, Panel C). rC/EBPβ-Asp¹⁰⁵ was not associated with active caspases 1 or 8 (data not shown). Similar to the effects of rC/EBPβ-PSer¹⁰⁵ and rC/EBPβ-Asp¹⁰⁵ on cell survival, treatment of freshly isolated stellate cells from C/EBPβ −/− mice with cell permeant tetrapeptides that include the phosphorylation mimic domain of C/EBPβ (Ac-KE²¹⁷VD-CHO) or rC/EBPβ (Ac-KKPD¹⁰⁵-CHO), rescued these cells from apoptosis following their activation by collagen type I (FIG. 7, Panel D). In addition, it was determined that the synthetic tetrapeptide Ac-KKPD¹⁰⁵-CHO, like Ac-KE²¹⁷VD-CHO, inhibited the in vitro self-activation of recombinant caspase 8 (FIG. 7, Panel E).

The results obtained during the development of the present invention indicate that the present invention provides a novel means for cell survival. However, an understanding of the mechanisms involved is not necessary in order to use the present invention. Nonetheless, signaling pathways that activate RSK result in the phosphorylation of C/EBPβ on Thr²¹⁷. C/EBPβ-PThr²¹⁷ associates with the initiator procaspases 1 and 8 and inhibits their processing, which blocks the apoptotic cascade and allows the survival of hepatic stellate cell upon activation. This is believed to be the first demonstration that phosphorylation of a transcription factor by the ERK/MAPK/RSK pathway stimulates its association with procaspases, preventing their activation. Phosphorylation of C/EBPβ on Thr²¹⁷ with the creation of a pseudo XEXD box (Thornberry et al., [1997], supra; and Blanchard et al., supra) provides the most likely explanation of this newly described anti-apoptotic role of this transcription factor. Although C/EBPβ Thr²¹⁷ associated with the initiator procaspases 1 and 8 (inhibiting their processing and activation), but not with the effector procaspases 3, 7 or 9 (Earnshaw et al., supra; Thornberry and Lazebnik, supra; and Ashkenazi and Dixit, supra), it effectively blocked the apoptotic cascade, as indicated by the lack of active caspase 3 in hepatic stellate cells from C/EBPβ^(+/+), but not from the nonphosphorylatable dominant negative C/EBPβ-Ala²¹⁷ transgenic, mice. Moreover, the phosphorylation mimic C/EBPβ-Glu²¹⁷ mutant rescued activated stellate cells from apoptosis induced either by expressing MEKK1, IκBα or RSK dominant negative mutants or by treating the cells with the proteasome inhibitor lactacystin (Chen et al., Nature 374:386-388 [1995b]; Lee et al., [1997], supra; and Nakajima et al., supra).

Neither the activation nor dimerization domains of C/EBPβ are indispensable for associating with procaspases 1 and 8 and inhibiting their processing. More importantly, the mouse Thr²¹⁷ phosphoacceptor is virtually identical in bovine and human C/EBPβ (Akira et al., supra; Cao et al., supra; and Yamaoka et al., supra). C/EBPβ-PThr²¹⁷ is mimicked by C/EBPβ-Glu²¹⁷ (KE²¹⁷VD) (Buck et al., [1999] supra) and therefore, conforms to the current knowledge about the structural requirements for caspase inhibitory tetrapeptides. These tetrapeptides require an aspartic acid (D) residue at the P1 position (Thornberry and Lazebnik, 1998), which is also present in C/EBPβ at amino acid D²¹⁹ (Cao et al., supra; and Buck et al., [1999], supra).

Using a positional scanning substrate library, Thornberry and coworkers (Thornberry et al., J. Biol. Chem., [1997]) have shown that although the optimal sequence for caspase 8 is I/L/V (P4 position) EXD, I, V, W, T, P and D are also acceptable at the P4 position, by virtue of being downstream cleavage sites in the apoptotic cascade. These findings have been recently corroborated by crystallography (Blanchard et al., supra). Group III caspases (including caspase 8) have a preference for small hydrophobic residues at P4, such as D used in these studies (Blanchard et al., supra), which has a comparable hydrophobicity to the K²¹⁶ in C/EBPβ (Boyle et al., Meth. Enzymol., 201:110-149 [1991]). In addition, these authors argue that subsite S3 of caspase 8, which interacts with position P3 (but not with P4) of the tetrapeptide, is crucial for determining the specificity, and that the original classification of caspases need to be revised, especially for caspase 8 (Blanchard et al., supra). Similarly to the selective effects of C/EBPβ PThr²¹⁷ shown herein, CrmA inhibits procaspases 1 and 8 selectively (K_(i)=0.01 and 0.95 nM, respectively) compared to procaspases 3, 6 and 7 (Zhou et al., J. Biol. Chem., 272:7797-7800 [1997]). However, other investigators have suggested that CrmA also inhibits caspases 4, 5, 9 and 10 (Garcia-Calvo et al., J. Biol. Chem., 273:32608-32613 [1998]). In addition, the baculovirus p35 is a potent, albeit less selective, inhibitor of procaspases 1, 3, 6, 8, and 10 (K_(i)=0.1 nM) (Andrade et al., Immunity 8:451-460 [1998]). Moreover, DEVD-CHO, which effectively inhibits procaspases 1 and 8 (K_(i)=17 and 12 nM) (Earnshaw et al., supra), is an equally potent inhibitor of caspase 8 as its classical inhibitor IETD (Blanchard et al., supra).

Additional support for the present invention is provided by the experimental data obtained during the development of the present invention and discussed herein. As indicated herein, the sequence KPhospho-T²¹⁷VD (or KE²¹⁷VD) within C/EBPβ is effective in associating with procaspases 1 and 8 and blocking their activation. Activation of hepatic stellate cells has been induced by treating animals with the hepatotoxin CCl₄, or by culturing these cells on a collagen type I matrix. The experiments with C/EBPβ −/− cells and with dominant negative Ala²¹⁷ and dominant positive Glu²¹⁷ mutants, including new C/EBPβ-Ala²¹⁷ transgenic mice, strongly support the present invention.

However, rat C/EBPβ contains a double mutation (mouse Thr-217->rat Ala-217 and mouse Ala-104->rat Ser-105) with a creation of a cell growth compensatory Ser105 phosphoacceptor (Buck et al., [1999], supra). Therefore, experiments were conducted in order to determine whether rat C/EBPβ-PSer¹⁰⁵ could play a comparable role to that of mouse C/EBPβ-PThr²¹⁷ on cell survival. Expression of the phosphorylation mimic rat C/EBPβ-Asp¹⁰⁵ (Trautwein et al., supra; and Buck et al., [1999], supra) was sufficient to rescue activated stellate from apoptosis induced by expressing a catalytically inactive dominant negative mutant RSK vector (Nakajima et al., supra; and Buck et al., [1999], supra), or by treating the cells with a proteasome inhibitor. Activation of the PKCα (Trautwein et al., supra) or MAPK (Nebreda and Gavin, supra; and Whitmarsh and Davis, [2000] supra) signaling pathways would result in phosphorylation of rC/EBPβ on Ser¹⁰⁵ (Trautwein et al., supra; and Buck et al., [1999], supra). It was found that the rat C/EBPβ-Ala¹⁰⁵ mutant behaves as a dominant negative, inducing apoptosis of activated mouse hepatic stellate cells.

The present invention, supported by experiments conducted on hepatic stellate cells are relevant to human diseases such as liver cirrhosis. In addition, it is contemplated that the present invention will find use in treatment of diseases related to tissue repair mechanisms, due to the activation of other mesenchymal cells (including, but not limited to brain, lung and kidney cells). For example, it is contemplated that the present invention will find use in treatment of such diseases as lung fibrosis (e.g., emphysema), brain gliosis (e.g., Alzheimer's disease), and kidney fibrosis (e.g., glomerulonephritis). However, it is not intended that the present invention be so limited. It is further contemplated that the creation of a pseudo XEXD box by phosphorylation, is a highly prevalent biological mechanism maintained through evolution. This is supported by the observation of potential pseudo XEXD boxes upon phosphorylation of threonine (Phospho-TVD, analogous to EVD) in more than 22,000 sites and of serine (EVPhospho-S, analogous to EVD) in more than 27,000 sites in a database containing ˜350,000 proteins. Thus, if only 1% of these sequences is phosphorylated in vivo it would generate pseudo XEXD boxes in ˜500 proteins.

Therapeutics

As discussed herein, it is contemplated that the C/EBPβ peptides of the present invention (e.g., mutant mouse, human and rat peptides) will find use in the therapy and prevention of diseases associated with abnormal fibrosis. Indeed, the modified C/EBPβs (referred to herein as “C/EBPβ”) find use in various settings involving the production of fibrosis. In one embodiment, C/EBPβ peptides are administered in combination with other conventional pharmaceutical agents. The combination of therapeutic agents having different mechanisms of action is contemplated to provide synergistic effects allowing for the use of lower effective doses of each agent and lessening side effects. In another embodiment, a vector expressing the polynucleotide encoding C/EBPβ may be administered to a subject to treat or prevent fibrosis. In still another embodiment, a vector expressing antisense of the polynucleotide encoding C/EBPβ may be administered to a subject to stimulate fibrosis (e.g., wound healing).

Expression vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue or cell population. In addition to the methods described elsewhere herein, methods which are well known to those skilled in the art can be used to construct recombinant vectors which will express C/EBPβ.

Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection and by liposome injections may be achieved using methods which are well known in the art.

Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administration of a pharmaceutical composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. Such pharmaceutical compositions may consist of C/EBPβ, antibodies to C/EBPβ, mimetics, agonists, antagonists, or inhibitors of C/EBPβ. The compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones. As indicated herein, the pharmaceutical compositions utilized in this invention may be administered by any number of suitable routes.

In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically, as known in the art.

Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient. Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

Pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are known to those in the art.

The pharmaceutical compositions of the present invention may be manufactured in a manner that is known in the art (e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes).

The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of C/EBPβ such labeling would include amount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.

For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays and/or in animal models. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information is then be used to determine useful doses and routes for administration in humans. A therapeutically effective dose refers to that amount of active ingredient which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., the ED50—the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.

Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.

Normal dosage amounts may vary depending upon the subject and the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to those skilled in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

Diagnosis

In another embodiment, antibodies which specifically bind C/EBPβ find use in the diagnosis of conditions or diseases characterized by increased or decreased fibrosis, or in assays to monitor patients being treated with C/EBPβ (e.g., the modified C/EBPβs of the present invention).

For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others, may be immunized by injection with C/EBPβ or any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially preferable.

Monoclonal antibodies to C/EBPβ may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture, including but not limited to the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique.

In addition, techniques developed for the production of “chimeric antibodies”, the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used, as known in the art. Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce C/EBPβ single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobin libraries, as known in the art. Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as known in the art.

Antibody fragments which contain specific binding sites for C/EBPβ may also be generated. For example, such fragments include, but are not limited to, the F(ab′)2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab′)2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity, as known in the art.

Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between C/EBPβ and its specific antibody.

Diagnostic assays for C/EBPβ include methods which utilize the antibody and a label to detect C/EBPβ in cells or tissues. The antibodies may be used with or without modification, and may be labeled by joining them, either covalently or non-covalently, with a reporter molecule. A wide variety of reporter molecules which are known in the art may be used, several of which are described above. A variety of protocols including ELISA, RIA, IFA, and FACS for measuring C/EBPβ are known in the art and provide one basis for monitoring levels of C/EBPβ expression.

In another embodiment of the invention, the polynucleotides encoding C/EBPβ are used for diagnostic purposes. The polynucleotides which may be used include oligonucleotide sequences, antisense RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of C/EBPβ may be correlated with disease and/or disease amelioration. The diagnostic assay may be used to distinguish between absence, presence, and excess expression of C/EBPβ, and to monitor regulation of C/EBPβ levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding C/EBPβ or closely related molecules, may be used to identify nucleic acid sequences which encode C/EBPβ. The specificity of the probe, whether it is made from a highly specific region and the stringency of the hybridization or amplification (maximal, high, intermediate, or low) will determine whether the probe identifies only naturally occurring sequences encoding C/EBPβ alleles, or related sequences (e.g., the modified C/EBPβs of the present invention).

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the following abbreviations apply: eq (equivalents); M (Molar); mM (millimolar); μM (micromolar); N (Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); g (grams); mg (milligrams); μg (micrograms); Kg (kilograms); L (liters); mL (milliliters); μL (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); h and hr (hours); min. (minutes); s and sec. (seconds); ° C. (degrees Centigrade); v/v (volume/volume); w/v (weight/volume), μCi (microcuries); FDA (United States Food and Drug Administration); DME (Dulbecco's Modified Eagles Medium); LDL (low-density lipoprotein); DMF (N,N-dimethylformamide); α-SMA (α-smooth muscle actin); MDA (malondialdehyde); 4-HNE (4-hydroxynonenal); PBS (phosphate buffered saline); FBS (fetal bovine serum); K₂CO₃ (potassium carbonate); NaHCO₃ (sodium bicarbonate); MgCl₂ (magnesium chloride); NaOH (sodium hydroxide); FeSO₄ (ferrous sulfate); MgSO₄ (magnesium sulfate); SD or S.D. (standard deviation); SEM (standard error of the mean); Accurate Chemical & Scientific Corp. (Westbury, N.Y.); Becton Dickinson (Hunt Valley, Md.); Amersham (Arlington Heights, Ill.); Charles River Breeding Labs (Wilmington, Mass.); Clonetics (Clonetics Corp., San Diego, Calif.); Pharmingen (San Diego, Calif.); Collaborative Bio-medical Products (Bedford, Mass.); DuPont (DuPont Co., Wilmington, Del.); Hitachi (Hitachi Scientific Instruments, Mountain View, Calif.); Hoechst (Santa Cruz Biotechnology, Santa Cruz, Calif.); Sigma (Sigma Chemical Company, St. Louis, Mo.); Molecular Probes (Eugene, Oreg.); Upstate Biotechnology (Lake Placid, N.Y.); Vector Laboratories (Burlingame, Calif.); and Alexis (San Diego, Calif.).

Unless otherwise indicated below, the results are expressed as the mean (±SEM) of at least triplicates, unless stated otherwise. Either the Student t of the Fisher's exact test was used to evaluate the differences in the means between groups, with a P value of <0.05 considered to be significant.

Various protein databases were searched during the development of the present invention, including the Atlas Retrieval System protein databases PIR1, PIR2, PIR3 and PATX containing more than 347,000 proteins, including more than 22,000 TVD and 27,000 EVS sequences. The proapoptotic mutants include mouse—alanine 217, human—alanine 266, and rat—alanine 105.

Example 1 Cell Cultures and Transfections

Adult male Sprague-Dawley rats and adult male C/EBPβ^(+/+) and C/EBPβ^(−/−) (Screpanti et al., supra; and Buck et al., [1999], supra), C/EBPβ-Ala²¹⁷ and C/EBPβ-Asp¹⁰⁵ transgenic mice were used for the isolation of primary hepatic stellate cells (Houglum et al., supra). Primary hepatic stellate cells were isolated by collagenase/pronase perfusion and purified by a single step density Nycodenz gradient (Accurate Chemical & Scientific) as known in the art (See, Lee et al., [1995], supra). Cells were plated on type I collagen matrix (Becton Dickinson) or EHS matrix (Matrigel, Collaborative Biomedical Products). Cells were cultured with 10% fetal calf serum/10% fetal bovine serum and transfected with lipofectamine (GIBCO) for DNA vectors or with the Chariot reagent (Active Motif) for peptides (10 μM) as known in the art (See, Houglum et al., supra; and Morris et al., J. Biol. Chem., 274:24942-24946 [1999]).

Transgenic mice expressing rat C/EBPβ-Asp¹⁰⁵ mutant or mouse C/EBPβ-Ala²¹⁷ mutant were generated using methods known in the art (See, Houglum et al., supra; and Lee et al., [1995], supra) and back crossed to FVB mice. The presence of the neo gene was used to identify the transgenic mice by Southern blots. Endogenously expressed transfected proteins were visualized using antibodies specific for HA or C/EBPβ (Santa Cruz Biotechnology).

Expression of C/EBPβ 1-253, with a deletion of the dimerization domain (Descombes et al., supra; Akira et al., supra) (FIG. 4, Panel A) and containing Thr²¹⁷ or Glu²¹⁷, but not Ala²¹⁷, was compatible with cell growth and survival. C/EBPβ 1-215 lacking the DNA binding and dimerization domains and the KT²¹⁷VD phosphoacceptor (See, FIG. 4, Panel A) had no effect on cell survival. In contrast, C/EBPβ 216-253-Ala²¹⁷, but not T²¹⁷ or Glu²¹⁷, lacking the activation and dimerization domains (See, FIG. 4, Panel A), stimulated apoptosis in control cells and following expression of wild type RSK (See, FIG. 4, Panel B). In addition, C/EBPβ 216-253-Glu²¹⁷ was sufficient to rescue cells from apoptosis induced by a catalytically inactive mutant RSK expression vector (RSK N′C′), containing point mutations in both ATP-binding domains (Nakajima et al., supra; and Buck et al., [1999], supra) (See, FIG. 4, Panel B). Using confocal microscopy, and in agreement with previous reports (Mao et al., supra; and Ritter et al., supra), procaspases 1 and 8 were detected in the nucleus and cytoplasm of activated stellate cells, which colocalized with C/EBPβ (See, FIG. 4, Panel C).

Immunoprecipitation of the C/EBPβ-HA and C/EBPβ 216-253-HA, with antibodies against HA, from activated stellate cells, showed that both the phosphorylatable Thr²¹⁷ and the phosphorylation mimic Glu²¹⁷, co-immunoprecipitated with procaspases 1 and 8 more efficiently than the nonphosphorylatable Ala²¹⁷ (See, FIG. 4, Panel D). Therefore, the C/EBPβ activation and dimerization domains (Descombes et al., supra; and Akira et al., supra) are not required for stellate cell survival or association with procaspases 1 and 8.

Example 2 Fluorescence Microscopy Methods

Fluorescent labels were observed using a triple-channel fluorescence microscope. The fluorochromes utilized were FITC and Texas red (Molecular Probes). The number of annexin V (+) cells was determined by in vivo microscopy as known in the art (See, Rudel and Bokoch, supra) among all cells or those expressing the GFP indicator protein either co-transfected with C/EBPβ proteins, or alone. These values are reported herein as a percentage of annexin V (+) for cells expressing the transfected DNA. At least 1000 cells were analyzed per experimental point, including at least 100 cells expressing the transfected DNA (Buck and Chojkier. EMBO J., 15:1753-1765 [1996]; Buck et al., [1994], supra; and Buck et al., [1999], supra). The ApoAlert mitochondrial-sensor assay (Clontech) used in some experiments utilizes a cationic dye that fluoresces red when aggregated within the mitochondria in healthy cells and green when it remains in monomeric form in the cytosol of apoptotic cells due to altered mitochondrial membrane potential (Green and Reed, supra). Nuclear morphology was analyzed by staining cells with Hoechst 33342 (Molecular Probes). Confocal microscopy was performed in stellate cells using antibodies specific for C/EBPβ and procaspase 8 (Santa Cruz Biotechnology).

For example, in one set of experiments, investigations were conducted in order to determine whether phosphorylation of Ser¹⁰⁵ confers to rC/EBPβ the ability to rescue cells from apoptosis. Activated mouse hepatic stellate cells cultured on a collagen type I matrix were induced to develop apoptosis either by expressing the dominant negative RSK mutant (Nakajima et al., supra) or by treating them with lactacystin (a proteasome inhibitor). Transfection of the phosphorylation mimic rC/EBPβ Asp¹⁰⁵ (Trautwein et al., supra), together with the transfection indicator CMV-GFP, rescued hepatic stellate cells from apoptosis as determined by the incorporation of annexin-V-PE using in vivo fluorescence microscopy (See, FIG. 5, Panel B). rC/EBPβ-Ala¹⁰⁵, like C/EBPβ-Ala²¹⁷, induced apoptosis of activated primary mouse hepatic stellate cells, since neither mutant has a RSK phosphoacceptor domain (Buck et al., [1999], supra). These results suggest that rC/EBPβ-PSer¹⁰⁵ (or rC/EBPβ-Asp¹⁰⁵), at least under the experimental conditions described, is able to block the apoptotic pathway as effectively as C/EBPβ-PThr²¹⁷ (See, FIG. 1).

Example 3 Animal Procedures and Testing

As described herein, investigations were conducted to determine whether hepatic stellate cell activation is stimulated by CCl₄, a hepatotoxin that induces hepatic oxidative stress and liver fibrogenesis (Houglum et al., supra; and Chojkier, supra). Transgenic mice expressing rat C/EBPβ-Asp¹⁰⁵ or mouse C/EBPβ-Ala²¹⁷ were generated as described herein, using methods known in the art (See, Houglum et al. [1995]. supra; and Lee et al. [1995], supra) and back-crossed to FVB mice. The presence of the neo gene was used to identify these transgenic mice by Southern blotting.

C/EBPβ^(+/+), C/EBPβ^(−/−) and C/EBPβ-Ala²¹⁷ or C/EBPβ-Asp¹⁰⁵ transgenic mice (25 g) each received a single intraperitoneal injection of CCl₄, in mineral oil (1:1, vol/vol) (50 μl) or mineral oil only (25 μl) (Buck et al., [1999], supra). Twelve hours later, animals were sacrificed and immunofluorescence analyzed by confocal microscopy (Buck et al., [1994], supra). Stellate cells were identified using antibodies specific for glial fibrillary acidic protein and vimentin (Santa Cruz Biotechnology). Active caspase 3 was observed using specific antibodies (Pharmingen).

Hepatic stellate cells, identified by their glial fibrillary acidic protein and vimentin staining (Ankoma-Sey and Friedman, supra), were induced to proliferate in the liver of C/EBPβ^(+/+) mice following treatment with a single dose of CCl₄. In contrast, CCl₄ induced stellate cell apoptosis in the livers of C/EBPβ^(−/−) and C/EBPβ-Ala²¹⁷ transgenic mice (See, FIG. 3), as determined by the presence of an active effector caspase 3. Because activated p90RSK is able to causing the in vitro phosphorylation of C/EBPβ on Thr²¹⁷ (Buck et al., [1999], supra), it was of interest to determine whether RSK was associated with C/EBPβ in these animals, as described in Example 4.

Example 4 Immunoprecipitation and Immunoblotting

C/EBPβ, procaspases 1 and 8 and RSK were detected by immunoblotting in cell and liver lysates (Buck et al., [1994], supra) following a chemiluminescence protocol (DuPont) using purified IgG antibodies, specific for C/EBPβ and RSK (Santa Cruz Biotechnology) and for procaspases 1 and 8 (Pharmingen). The results were confirmed using rabbit anti-RSK-5 serum (kindly provided by J. Blenis) and anti-RSK R23820 (Transduction Laboratories).

Because activated p90RSK is able to cause the in vitro phosphorylation of C/EBPβ on Thr²¹⁷ (Buck et al., [1999], supra), it was of interest to determine whether RSK was associated with C/EBPβ in these animals. In these experiments, C/EBPβ was immunoprecipitated with specific antibodies from primary hepatic stellate cells freshly isolated from C/EBPβ^(+/+) and C/EBPβ-Ala²¹⁷ mice treated with CCl₄. The results indicated that there was an increased RSK association with C/EBPβ in stellate cells isolated from C/EBPβ^(+/+), but not C/EBPβ-Ala²¹⁷ mice treated with CCl₄, as indicated in FIG. 3. RSK was identified using specific antibodies against pp90RSK (Santa Cruz Biotechnology), which do not cross-react with p70 S6 kinase. These results were confirmed using two other specific antibodies, anti-RSK-5 (kindly provided by J. Blenis) and anti-RSK (Transduction Laboratories). When RSK protein was immunoprecipitated with specific antibodies, C/EBPβ was found to co-immunoprecipitate, and the level of coprecipitated C/EBPβ was consistently increased in C/EBPβ^(+/+) mice treated with CCl₄. Control preimmune serum, anti-β-galactosidase, or purified IgG yielded immunoprecipitates containing undetectable levels of either RSK of C/EBPβ, confirming the specificity of the co-immunoprecipitation procedure. These findings strongly suggest that phosphorylation of C/EBPβ on Thr²¹⁷ by RSK is indispensable for hepatic stellate cell survival.

Example 5 Phosphorylation of C/EBPβ

Day-4 primary mouse or rat hepatic stellate cells cultured on an EHS (Matrigel; Becton-Dickinson) (quiescent cells) of collagen type I (Collaborative Research) (activated cells) were labeled with 30 mCi ³²P-orthophosphate for 12 hours (cultured for the last 30 min with 20% fetal calf serum) and two-dimensional tryptic maps were performed as known in the art (See, Trautwein et al., supra; Buck et al., [1999], supra).

For RSK phosphorylation reactions in vitro, 3 μg of immunopurified C/EBPβ, C/EBPβ-Ala²¹⁷, rC/EBPβ and rC/EBPβ-Ala¹⁰⁵ were assayed as known in the art (See, Buck et al. [1999], supra). The identity of the phosphopeptides containing PThr²¹⁷ or PSer¹⁰⁵ was suggested by the migration of the putative peptide and confirmed with specific antibodies against the phosphorylated epitopes of human (KSKAKK-PhosphoT²⁶⁶VDKHSD [SEQ ID NO:16]; homologous to mouse PThr²¹⁷) and rat (KPSKKP-PhosphoS¹⁰⁵DYGYVS [SEQ ID NO:17]) C/EBPβ.

In experiments to test whether collagen type I matrix induces C/EBPβ phosphorylation, primary mouse hepatic stellate cells were labeled for 12 hr with ³²P-orthophosphate and cultured either on EHS (Matrigel) (quiescent cells) or collagen type I (activated cells). Phosphopeptide mapping of immunoprecipitated C/EBPβ showed that collagen type I matrix induced a site-specific phosphorylation of endogenous C/EBPβ on Thr²¹⁷, a p90RSK phosphoacceptor domain (Buck et al., [1999], supra), and on another, yet unidentified, phosphoacceptor site, as indicated by the results in FIG. 1. C/EBPβ phosphorylation was negligible in quiescent mouse stellate cells cultured on an EHS matrix (See, FIG. 1).

Although the C/EBPβ-Thr²¹⁷ phosphoacceptor is highly conserved through evolution including human C/EBPβ, (Cao et al., supra; Akira et al., supra; and Yamaoka et al., supra), rat (r)C/EBPβ has evolved with a double mutation, lacking the Thr phosphoacceptor but having a compensatory Ser¹⁰⁵ phosphoacceptor (Descombes et al., supra; and Poli et al., supra). C/EBPβ-PThr²¹⁷ or rC/EBPβ-PSer¹⁰⁵ are critical for extracellular factor-induced cell proliferation (Buck et al., [1999], supra). To test whether collagen type I matrix induces rC/EBPβ phosphorylation, primary rat hepatic stellate cells were labeled for 12 hr with ³²P-orthophosphate and cultured either on EHS (Matrigel) (quiescent cells) or collagen type I (activated cells). Phosphopeptide mapping of immunoprecipitated C/EBPβ showed that collagen type I matrix induced phosphorylation of endogenous rC/EBPβ on Ser¹⁰⁵, a p90RSK phosphoacceptor domain (Buck et al., [1999], supra), and on other, yet unidentified phosphoacceptor sites (See, FIG. 6, Panel A). rC/EBPβ phosphorylation was negligible in quiescent rat stellate cells cultured on an EHS matrix (See, FIG. 6, Panel A).

Next, the functional relevance of C/EBPβ and its phosphorylation on Thr²¹⁷ was ascertained. Activated primary mouse hepatic stellate cells were transfected with either wild-type (sense) or antisense C/EBPβ expressing vectors. Stellate cells expressing C/EBPβ antisense, but not sense, RNA (Buck et al., [1994], supra) did not proliferate, as detected by the expression of proliferating cell nuclear antigen (Bravo et al., supra; Buck et al., [1994], supra; and Buck et al., [1999], supra), but displayed increased annexin-V binding to phosphatidylserine in plasma membranes, an early indicator of apoptosis (Rudel and Bokoch, supra) (See, FIG. 1, Panel C). To determine whether phosphorylation of mouse C/EBPβ on Thr²¹⁷ is critical for collagen type 1-induced proliferation of mouse hepatic stellate cells, cells were transfected with a MSV expression vector for the nonphosphorylatable C/EBPβ-Ala²¹⁷ mutant. Transfected cells were identified by the expression of the co-transfected CMV-GFP (green fluorescent protein) using triple-channel fluorescence microscopy. Cells expressing the C/EBPβ-Ala²¹⁷ mutant (Buck et al., [1999], supra) did not proliferate and became apoptotic as determined by live microscopy (FIG. 1, Panel C) and cell sorting for the transfection indicator GFP and annexin-V. Conversely, induction of apoptosis in hepatic stellate cells by blocking activation of the antiapoptotic NFκB (Beg and Baltimore, supra; and Wang et al., supra) or RSK (Buck et al., [1999], supra; Bonni et al., supra; Bhatt and Ferrell, supra; Gross et al., supra; and Sassone-Corsi et al., supra) was rescued by the phosphorylation mimic C/EBPβ-Glu²¹⁷ mutant (Buck et al., [1999], supra), as indicated in FIG. 1, Panel D.

MEK kinase 1 (MEKK1) activates NFκB through phosphorylation of IκB kinases α and β leading to phosphorylation of IκB (Lee et al., [1997], supra; Nakano et al., supra), which undergoes rapid proteolysis via the ubiquitin-proteasome pathway (Chen et al., [1995a], supra). Therefore, activated primary stellate cells were transfected with expression vectors for the dominant negative mutants for MEKK1, IκBα and RSK, or treated cells with lactacystin (a proteasome inhibitor) to induce apoptosis. In each case, stellate cell apoptosis was rescued by expressing C/EBPβ-Glu²¹⁷ (See, FIG. 1, Panel D), suggesting that in the absence of active NFκB or RSK, phosphorylation of C/EBPβ on Thr²¹⁷ is sufficient for hepatic stellate cell survival.

To ascertain the functional relevance of mouse C/EBPβ, and its phosphorylation on Thr²¹⁷, the survival of primary hepatic stellate cells isolated from mice with a targeted deletion of C/EBPβ (Screpanti et al., supra) was also investigated. C/EBPβ^(−/−), but not C/EBPβ^(+/+), stellate cells were refractory to growth stimulation by collagen type I and became apoptotic as determined by annexin-V binding using live microscopy (FIG. 2, Panels A and C). To study the role of C/EBPβ-PThr²¹⁷ on stellate cell survival, mice expressing a nonphosphorylatable dominant negative C/EBPβ-Ala²¹⁷ mutant transgene were developed. Although C/EBPβ-Ala²¹⁷ transgenic mice were developmentally normal, hepatic stellate cells isolated from these animals consistently failed to survive when activated by collagen type I (See, FIG. 2, Panels A and C). Apoptosis of stellate cells isolated from C/EBPβ^(−/−) and C/EBPβ-Ala²¹⁷ mice was corroborated by an increase in mitochondrial membrane permeability, using a mitochondrial sensor assay (See, FIG. 2, Panel C).

Example 6 Induction of Mesenchymal Cell Apoptosis by C/EBPβ-Ala²¹⁷

In these experiments, C/EBPβ-Ala²¹⁷ was tested in two mesenchymal cell lines for its ability to induce apoptosis. Human mesenchymal cell lines 1321-N1 (glial cells; obtained from Dr. Joan Brown, at the University of California, San Diego), RD (skeletal muscle cells; ATCC Accession No. CCL-136), and human fibroblasts (98VA3N cells, obtained from Dr. Jerry VanderBerg, at the Veterans Administration Medical Center, San Diego, Calif.) were cultured in minimal essential medium supplemented with fetal bovine serum as known in the art (See e.g., Buck et al., EMBO J., 13:151-160 [1994]). As additional cell types find use in the present invention, it is not intended that the present invention be limited to these specific cell lines. Indeed, similar results have been obtained with other cell types (e.g., CCF-STTG1 cells [ATCC Accession No. CRL-1718]) (data not shown).

Cells were co-transfected with vectors expressing the indicator protein GFP and C/EBPβ-Ala²¹⁷ as known in the art (See, Buck et al., Mol. Cell., 4:1087-1092 [1999]). Control cells were only transfected with GFP. Four hours after transfection, cells were assayed for annexin-V binding to exposed phosphatidylserine in plasma membranes, an early indicator of apoptosis, using live microscopy as known in the art, using a commercially available apoptosis kit (R & D Systems) according to the manufacturer's instructions (See also, Rudel and Bokoch, Science 276:1571-1574 [1997]).

Expression of the non-phosphorylatable C/EBPβ-Ala²¹⁷ mutant induced apoptosis of these mesenchymal cell lines, as indicated in Table 1, below.

TABLE 1 Induction of Mesenchymal Cell Apoptosis by C/EBPβ-Ala²¹⁷ Apoptosis (%) Cell Line Control C/ERPβ-Ala²¹⁷ RD 9 46* 1321 NI 4 56* 98VA3N 6 80* *P < 0.01

Thus, these data indicate that C/EBPβ-Ala²¹⁷ will find use as a therapeutic in the treatment of fibrosis in various tissues and cell types.

Example 7 Procaspase Activation and Caspase Activity

In these experiments, purified recombinant and synthetic C/EBPβ peptides were assayed for their ability to inhibit the activation of purified recombinant procaspases 1 and 8 (Alexis Biochemicals).

Purified recombinant C/EBPβ peptides, expressed as known in the art (See, Descombes et al., supra; and Buck et al. [1994], supra), and purified synthetic N-acetyl, C-aldehyde tetrapeptides (200 μM) (American Peptide Company) were assayed for their ability to inhibit the activity of purified human recombinant caspases 1 (catalogue #201-056) and 8 (catalogue #201-041-C005) (Alexis Biochemicals). The sequence of caspase 8 includes Ser217 through Asp479 cloned into an expression vector containing a 21 amino acid linker at the N-terminus. Thus, the prodomain (first 220 amino acids) is essentially missing. The fragment is cleaved at Asp385 and the active caspases are essentially identical to those identified in apoptotic cells (Stennicke and Salvesen, Meth. Enzymol., 322:91-100 [1997]). Caspase activity was determined by the release of the p-nitroanaline colorimetric (Alexis Biochemicals) substrate for caspase I (catalogue #260-026) or 8 (catalogue #260-045) within the linear part of the kinetic assay (Thornberry et al. [1997], supra; Stennicke and Salvesen, supra). Caspase 1 and 8 activity in cells was performed with colorimetric kits (catalogues #850-211-K and 850-221-K) as described by the manufacturer (Alexis) (See also, Thornberry et al. [1997], supra). The caspase 8 inhibitor IETD was from Calbiochem (Catalogue #218773).

Given that the Thr²¹⁷ phosphoacceptor in C/EBPβ contains a KT²¹⁷VD sequence to bovine and human C/EBPβ (Buck et al., [1999], supra), that upon phosphorylation by RSK can be functionally mimicked by the C/EBPβ-Glu²¹⁷ sequence KE²¹⁷VD (Buck et al., [1999], supra), and that the latter is similar to a XEXD box, found in inhibitors and substrates of caspases (Thornberry et al., supra; and Blanchard et al., supra), experiments were conducted to investigate whether C/EBPβ-PThr²¹⁷ associates with procaspases. C/EBPβ was immunoprecipitated using specific antibodies, from freshly isolated hepatic stellate cells from C/EBPβ^(+/+) and C/EBPβ-Ala²¹⁷ transgenic mice. With the use of specific antibodies, the initiator procaspases 1 and 8 were identified (See, FIG. 3, Panel D) but not the effector procaspases 3, 7 or 9 (Thornberry and Lazebnik, supra; Ashkenazi and Dixit, supra; and Earnshaw et al., supra), in C/EBPβ but not in control IgG, immunoprecipitates of activated C/EBPβ^(+/+) stellate cells, following treatment with CCl₄.

The association of C/EBPβ with procaspases 1 and 8 remained baseline in cells from C/EBPβ-Ala²¹⁷ mice treated with CCl₄ (FIG. 3), as well as in cells from C/EBPβ^(+/+) mice treated with control mineral oil. In addition, the activities of the initiator caspases 1 and 8 (See, FIG. 3), as well as that of the effector caspase 3 (See, FIG. 3), were increased in stellate cells from C/EBPβ^(−/−) or C/EBPβ-Ala²¹⁷, but not from C/EBPβ^(+/+) mice following treatment of these animals with CCl₄. These results indicate that phosphorylation of C/EBPβ on Thr²¹⁷ or the phosphorylation mimic C/EBPβ-Glu²¹⁷ mutant are required for C/EBPβ to associate with procaspases 1 and 8, and suggest that they may prevent processing and activation of procaspases 1 and 8, since neither KPhospho-T²¹⁷VD nor KE²¹⁷VD are the preferred substrates for caspases 1 or 8 (Wilson et al., supra; Margolin et al., supra; and Earnshaw et al., supra).

To delineate the role of C/EBPβ on the activation of procaspases 1 and 8, the self-activation of recombinant procaspases 1 and 8 was induced in vitro (Wilson et al., supra; Margolin et al., supra; and Earnshaw et al., supra), and the effect of purified recombinant C/EBPβ peptides, produced as described previously (Descombes et al., supra; and Buck et al., [1994], supra) on this process was determined. Consistent with the in vivo results described above, C/EBPβ-HA-Glu²¹⁷ and C/EBPβ 216-253-HA-Glu²¹⁷, but not C/EBPβ-HA-Ala²¹⁷, peptides associated with procaspases 1 and 8 (See, FIG. 5, Panel A). Recombinant C/EBPβ-Glu²¹⁷, and the synthetic peptides Ac-KPhospho-T²¹⁷VD-CHO and Ac-KE²¹⁷VD-CHO inhibited activation of procaspases 1 and 8 (FIG. 5, Panel B). In addition, in activated hepatic stellate cells, the cell permeant peptide Ac-KA²¹⁷VD-CHO induced apoptosis (See, FIG. 5, Panel C).

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art and/or related fields are intended to be within the scope of the following claims. 

1. A method for inducing apoptosis comprising administering a composition to hepatic stellate cells, wherein said composition comprises N-acetyl-KAVD-C-aldehyde peptide (Ac-KAVD-CHO) consisting of residues 216-219 of SEQ ID NO:3 or N-acetyl-KKPA-C-aldehyde peptide (Ac-KKPA-CHO) consisting of residues 102-105 of SEQ ID NO:14.
 2. The method of claim 1, wherein said composition comprises said N-acetyl-KAVD-C-aldehyde peptide (Ac-KAVD-CHO) consisting of residues 216-219 of SEQ ID NO:3.
 3. The method of claim 1, wherein said composition comprises said N-acetyl-KKPA-C-aldehyde peptide (Ac-KKPA-CHO) consisting of residues 102-105 of SEQ ID NO:14.
 4. The method of claim 1, wherein said hepatic stellate cells are within a subject, and wherein said administering is under conditions such that the endogenous phosphopeptides of said subject inhibit the activation of at least one caspase of said subject.
 5. The method of claim 4, wherein said subject is a mammal.
 6. The method of claim 5, wherein said mammal is a human.
 7. The method of claim 5, wherein said mammal is suffering from a fibrosis-related disease, wherein said fibrosis-related disease is hepatic disease.
 8. The method of claim 7, wherein said administration diminishes fibrosis.
 9. The method of claim 7, wherein said administration ameliorates fibrosis. 